Interferon Alpha Antibodies And Their Uses

ABSTRACT

The present invention provides isolated anti-interferon alpha monoclonal antibodies, particularly human monoclonal antibodies, that inhibit the biological activity of multiple interferon (IFN) alpha subtypes but do not substantially inhibit the biological activity of IFN alpha 21 or the biological activity of either IFN beta or IFN omega. Immunoconjugates, bispecific molecules and pharmaceutical compositions comprising the antibodies of the invention are also provided. The invention also provides methods for inhibiting the biological activity of IFN alpha using the antibodies of the invention, as well as methods of treating disease or disorders mediated by IFN alpha, such as autoimmune diseases, transplant rejection and graft versus host disease, by administering the antibodies of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 12/551,250, filed Aug. 31, 2009, which is adivisional application of U.S. patent application Ser. No. 11/009,410,filed Dec. 10, 2004, now U.S. Pat. No. 7,741,449, which claims priorityto U.S. Provisional Appl. Ser. No. 60/528,757, filed Dec. 10, 2003, thecontents of which are incorporated by reference in their entirety.

SEQUENCE LISTING

The specification further incorporates by reference the Sequence Listingsubmitted via EFS on Aug. 16, 2011. The Sequence Listing text file,identified as 077375.0931SEQLIST.txt, is 27,595 bytes and was createdAug. 16, 2011. The Sequence Listing, electronically filed, does notextend beyond the scope of the specification and does not contain newmatter.

BACKGROUND OF THE INVENTION

Type I interferons (IFN) (IFN-α, IFN-β, IFN-ω, IFN-τ) are a family ofstructurally related cytokines having antiviral, antitumor andimmunomodulatory effects (Hardy et al. (2001) Blood 97:473; Cutrone andLanger (2001) J. Biol. Chem. 276:17140). The human IFNα locus includestwo subfamilies. The first subfamily consists of at least 14 non allelicgenes and 4 pseudogenes having at least 75% homology. The secondsubfamily, αII or omega (ω), contains 5 pseudogenes and 1 functionalgene which exhibits 70% homology with the IFNα genes. The subtypes ofIFNα have different specific activities but they possess the samebiological spectrum (Streuli et al. (1981) Proc. Natl. Acad. Sci. USA78:2848) and have the same cellular receptor (Agnet M. et al. (1983) in“Interferon 5” Ed. I. Gresser p. 1-22, Academic Press, London).

All human type I interferons bind to a cell surface receptor (IFN alphareceptor, IFNAR) consisting of two transmembrane proteins, IFNAR-1 andIFNAR-2 (Uze et. al. (1990) Cell 60:225; Novick et al. (1994) Cell77:391; Pestka et al. (1987) Annu Rev. Biochem. 56:727; Mogensen et al.(1999) J. Interferon Cytokine Res. 19:1069). IFNAR-1 is essential forhigh affinity binding and differential specificity of the IFNAR complex(Cutrone (2001) supra). While functional differences for each of thetype I IFN subtypes have not been identified it is thought that each mayexhibit different interactions with the IFNAR receptor componentsleading to potentially diverse signaling outcomes (Cook et al. (1996) J.Biol. Chem. 271:13448). In particular, studies utilizing mutant forms ofIFNAR1 and IFNAR2 suggested that alpha and beta interferons signaldifferently through the receptor by interacting differentially withrespective chains (Lewerenz et al (1998) J. Mol. Biol. 282:585).

Early functional studies of type I IFNs focused on innate defenseagainst viral infections (Haller et al. (1981) J. Exp. Med. 154:199;Lindenmann et al (1981) Methods Enzymol. 78:181). More recent studies,however, implicate type I IFNs as potent immunoregulatory cytokines inthe adaptive immune response. Specifically, type I IFNs have been shownto facilitate differentiation of naïve T cells along the Th1 pathway(Brinkmann et al. (1993) J. Exp. Med. 178:1655), to enhance antibodyproduction (Finkelman et al. (1991) J. Exp. Med. 174:1179) and tosupport the functional activity and survival of memory T cells (Santini,et al. (2000) J. Exp. Med. 191:1777; Tough et al. (1996) Science272:1947).

Recent work by a number of groups suggests that IFN-α may enhance thematuration or activation of dendritic cells (DCs) (Santini, et al.(2000) J. Exp. Med. 191:1777; Luft et al. (1998) J. Immunol. 161:1947;Luft et al. (2002) Int. Immunol. 14:367; Radvanyi et al. (1999) Scand.J. Immunol. 50:499; Paquette et al (1998) J. Leukoc. Biol. 64:358).Furthermore, increased expression of type I interferons has beendescribed in numerous autoimmune diseases (Foulis et al. (1987) Lancet2:1423; Hooks et al (1982) Arthritis Rheum 25:396; Hertzog et al. (1988)Clin. Immunol. Immunopathol. 48:192; Hopkins and Meager (1988) Clin.Exp. Immunol. 73:88; Arvin and Miller (1984) Arthritis Rheum. 27:582).The most studied examples of this are insulin-dependent diabetesmellitus (IDDM) (Foulis (1987) supra), systemic lupus erythematosus(SLE) (Hooks (1982) supra; Blanco et al (2001) Science 294:1540;Ytterberg and Schnitzer (1982) Arthritis Rheum. 25:401; Batteux et al.(1999) Eur. Cytokine Netw. :509), and autoimmune thyroiditis (Prummeland Laurberg (2003) Thyroid 13:547; Mazziotti et al. (2002) J.Endocrinol. Invest. 25:624; You et al. (1999) Chin. Med. J. 112:61; Kohet al. (1997) Thyroid 7:891), which are all associated with elevatedlevels of IFN α, and rheumatoid arthritis (RA) (Hertzog (1988), Hopkinsand Meager (1988), Arvin and Miller (1984), supra) in which IFN-β mayplay a more significant role.

Moreover, administration of interferon α has been reported to exacerbateunderlying disease in patients with psoriasis, autoimmune thyroiditisand multiple sclerosis and to induce an SLE like syndrome in patientswithout a previous history of autoimmune disease. Interferon α has alsobeen shown to induce glomerulonephritis in normal mice and to acceleratethe onset of the spontaneous autoimmune disease of NZB/W mice. Further,IFN-α therapy has been shown in some cases to lead to undesired sideeffects, including fever and neurological disorders. Hence, there arepathological situations in which inhibition of IFN-α activity may bebeneficial to the patient and a need exists for agents effective ininhibiting IFN-α activity.

SUMMARY OF THE INVENTION

The present invention provides isolated monoclonal antibodies that bindto IFN alpha and inhibit the biological activity of multiple IFN alphasubtypes, but not substantially inhibit the biological activity of IFNalpha subtype 21, or of IFN beta or IFN omega. In preferred embodiments,the antibodies of the invention are capable of inhibiting surfaceexpression of cell markers induced by IFN alpha, inhibiting IP-10expression induced by IFN alpha and/or inhibiting dendritic celldevelopment mediated by plasma from patients with systemic lupuserythematosus (SLE). These antibodies can be used for therapeutic,including prophylactic, purposes, for example in situations where theproduction or expression of interferon alpha is associated withpathological symptoms. Such antibodies can also be used for thediagnosis of various diseases or for the study of the evolution of suchdiseases.

In one embodiment, the present invention includes an antibody orantibody fragment that binds to IFN alpha, preferably human IFN alpha(e.g., human IFN alpha 2a, human IFN alpha 2b), and inhibits thebiological activity of multiple IFN alpha subtypes, but does notsubstantially inhibit the biological activity of IFN alpha subtype 21,or IFN beta or IFN omega. In addition, in various embodiments, theantibodies of the invention are capable of inhibiting surface expressionof cell markers induced by IFN alpha, inhibiting IP-10 expressioninduced by IFN alpha and/or inhibiting dendritic cell developmentmediated by plasma from patients with systemic lupus erythematosus(SLE). The antibody or antibody fragment preferably is a human antibodyor antibody fragment, or alternatively can be a murine, chimeric orhumanized antibody. In certain embodiments, an antibody of the inventionfunctions by a non-competitive mechanism of action. For example, inpreferred embodiments, the antibody: (i) does not inhibit the binding ofan IFN alpha, such as IFN alpha 2a, to cells expressing interferon alphareceptor (IFNAR) and (ii) binds to cells expressing IFNAR in thepresence of an IFN alpha, such as IFN alpha 2a.

In one aspect, the invention pertains to isolated antibodies, or antigenbinding portions thereof, wherein the antibodies:

(a) comprise a heavy chain variable region of a human VH 1-18 or 4-61gene;

(b) comprise a light chain variable region of a human A27 gene; and

(c) inhibit the biological activity of interferon alpha (e.g., inhibitsthe biological activity of at least one IFN alpha subtype).

In another aspect, the invention pertains to isolated monoclonalantibodies, or antigen binding portions thereof, comprising a heavychain variable region comprising CDR1, CDR2, and CDR3 sequences and alight chain variable region comprising CDR1, CDR2, and CDR3 sequences,wherein:

(a) the heavy chain variable region CDR3 sequence comprises the aminoacid sequence of SEQ ID NO: 7, 8, or 9, or conservative modificationsthereof;

(b) the light chain variable region CDR3 sequence comprises the aminoacid sequence of SEQ ID NO: 16, 17, or 18, or conservative modificationsthereof;

(c) the antibody inhibits the biological activity of multiple IFN alphasubtypes but does not substantially inhibit the biological activity ofIFN alpha 21; and

(d) the antibody exhibits at least one of the following properties:

-   -   (i) the antibody does not substantially inhibit the biological        activity of IFN beta or IFN omega;    -   (ii) the antibody inhibits IFN-induced surface expression of        CD38 or MHC Class I on peripheral blood mononuclear cells;    -   (iii) the antibody inhibits IFN-induced expression of IP-10 by        peripheral blood mononuclear cells;    -   (iv) the antibody inhibits dendritic cell development mediated        by systemic lupus erythematosus (SLE) plasma.

In such antibodies, the heavy chain variable region CDR2 sequence cancomprise the amino acid sequence of SEQ ID NO: 4, 5, or 6, orconservative modifications thereof; and the light chain variable regionCDR2 sequence can comprise the amino acid sequence of SEQ ID NO: 13, 14,or 15, or conservative modifications thereof. Furthermore, in suchantibodies, the heavy chain variable region CDR1 sequence can comprisethe amino acid sequence of SEQ ID NO: 1, 2, or 3, or conservativemodifications thereof; and the light chain variable region CDR1 sequencecan comprise the amino acid sequence of SEQ ID NO: 10, 11, or 12, orconservative modifications thereof.

In another aspect, the invention pertains to isolated monoclonalantibodies, or antigen binding portions thereof, comprising a heavychain variable region and a light chain variable region, wherein:

(a) the heavy chain variable region comprises an amino acid sequencethat is at least 80% homologous to SEQ ID NO: 19, 20, or 21;

(b) the light chain variable region comprises an amino acid sequencethat is at least 80% homologous to SEQ ID NO: 22, 23, or 24;

(c) the antibody inhibits the biological activity of multiple IFN alphasubtypes but does not substantially inhibit the biological activity ofIFN alpha 21; and

(d) the antibody exhibits at least one of the following properties:

-   -   (i) the antibody does not substantially inhibit the biological        activity of IFN beta or IFN omega;    -   (ii) the antibody inhibits IFN-induced surface expression of        CD38 or MHC Class I on peripheral blood mononuclear cells;    -   (iii) the antibody inhibits IFN-induced expression of IP-10 by        peripheral blood mononuclear cells;    -   (iv) the antibody inhibits dendritic cell development mediated        by systemic lupus erythematosus (SLE) plasma.

In another aspect, the invention pertains to isolated monoclonalantibodies, or antigen binding portions thereof, comprising a heavychain variable region and a light chain variable region, wherein:

(a) the heavy chain variable region comprises an amino acid sequencecomprising the amino acid sequence of SEQ ID NO: 19, 20, or 21; and

(b) the light chain variable region comprises an amino acid comprisingthe amino acid sequence of SEQ ID NO: 22, 23, or 24;

wherein the antibody inhibits the biological activity of interferonalpha (e.g., inhibits the biological activity of at least one IFN alphasubtype).

In yet another aspect, the invention pertains to mutated variants of SEQID NO: 19 having increased stability. Preferred embodiments include anisolated monoclonal antibody, or antigen binding portion thereofcomprising:

-   -   (a) a heavy chain variable region comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 34,        35, 36 and 37; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO: 22;    -   wherein the antibody inhibits the biological activity of at        least one interferon alpha subtype.

In yet another aspect, the invention pertains to an isolated monoclonalantibody, or antigen binding portion thereof comprising:

-   -   (a) a heavy chain variable region CDR1 comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 1, 2        and 3;    -   (b) a heavy chain variable region CDR2 comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 4, 5        and 6;    -   (c) a heavy chain variable region CDR3 comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 7, 8        and 9;    -   (d) a light chain variable region CDR1 comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 10,        11 and 12;    -   (e) a light chain variable region CDR2 comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 13,        14 and 15; and    -   (f) a light chain variable region CDR3 comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 16,        17 and 18;    -   wherein the antibody inhibits the biological activity of        interferon alpha (e.g., inhibits the biological activity of at        least one IFN alpha subtype).

In yet another aspect, the invention pertains to an isolated monoclonalantibody, or antigen binding portion thereof, that competes for bindingto IFN alpha 2a or IFN alpha 2b with any of the above mentionedantibodies.

In yet another aspect, the invention pertains to an isolated humanantibody, or antigen-binding portion thereof, that inhibits thebiological activity of multiple interferon (IFN) alpha subtypes, whereinthe antibody does not inhibit binding of IFN alpha to interferon alphareceptor (IFNAR)-expressing cells and wherein the antibody associateswith IFNAR-expressing cells in the presence, but not the absence, of IFNalpha.

The invention also encompasses nucleic acid molecules that encode theantibodies or antigen-binding portions thereof in any of the abovementioned antibodies.

The antibodies of the invention can be of any isotype. Preferredantibodies are of the IgG1 or IgG4 isotype. The antibodies of theinvention can be full-length antibodies comprising variable and constantregions, or they can be antigen-binding fragments thereof, such as asingle chain antibody or a Fab fragment.

The invention also encompasses immunoconjugates of the antibodies of theinvention, in which the antibody is linked to a therapeutic agent, suchas a cytotoxin or a radioactive isotope. The invention also encompassesbispecific molecules comprising an antibody of the invention, in whichthe antibody is linked to a second functional moiety having a differentbinding specificity than the antibody.

Pharmaceutical compositions comprising an antibody, or antigen bindingportion thereof, or immunoconjugate or bispecific molecule thereof, arealso provided. Such pharmaceutical compositions comprise the activeagent and a pharmaceutically acceptable carrier.

In another aspect, the present invention includes a method of inhibitingthe biological activity of interferon alpha, either in vivo or in vitro,comprising contacting interferon alpha with an anti-IFN alpha antibodyof the invention, such that the biological activity of interferon alphais inhibited.

In another aspect, the present invention includes a method of treatingan interferon alpha-mediated disease or disorder in a subject,comprising administering to the subject an anti-IFN alpha antibody ofthe invention, such that the interferon-alpha mediated disease in thesubject is treated. Examples of diseases that can be treated includeautoimmune diseases (e.g., systemic lupus erythematosus, multiplesclerosis, insulin dependent diabetes mellitus, inflammatory boweldisease, psoriasis, autoimmune thyroiditis, rheumatoid arthritis andglomerulonephritis), transplant rejection and graft versus host disease.

Other features and advantages of the instant invention will be apparentfrom the following detailed description and examples, which should notbe construed as limiting. The contents of all references, patents andpublished patent applications cited throughout this application areexpressly incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the nucleotide sequence (SEQ ID NO: 25) and amino acidsequence (SEQ ID NO: 19) of the heavy chain variable region of the 13H5human monoclonal antibody. The CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 4)and CDR3 (SEQ ID NO: 7) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 1B shows the nucleotide sequence (SEQ ID NO: 28) and amino acidsequence (SEQ ID NO: 22) of the light chain variable region of the 13H5human monoclonal antibody. The CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO:13) and CDR3 (SEQ ID NO: 16) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 2A shows the nucleotide sequence (SEQ ID NO: 26) and amino acidsequence (SEQ ID NO: 20) of the heavy chain variable region of the 13H7human monoclonal antibody. The CDR1 (SEQ ID NO: 2), CDR2 (SEQ ID NO: 5)and CDR3 (SEQ ID NO: 8) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 2B shows the nucleotide sequence (SEQ ID NO: 29) and amino acidsequence (SEQ ID NO: 23) of the light chain variable region of the 13H7human monoclonal antibody. The CDR1 (SEQ ID NO: 11), CDR2 (SEQ ID NO:14) and CDR3 (SEQ ID NO: 17) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 3A shows the nucleotide sequence (SEQ ID NO: 27) and amino acidsequence (SEQ ID NO: 21) of the heavy chain variable region of the 7H9human monoclonal antibody. The CDR1 (SEQ ID NO: 3), CDR2 (SEQ ID NO: 6)and CDR3 (SEQ ID NO: 9) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 3B shows the nucleotide sequence (SEQ ID NO: 30) and amino acidsequence (SEQ ID NO: 24) of the light chain variable region of the 7H9human monoclonal antibody. The CDR1 (SEQ ID NO: 12), CDR2 (SEQ ID NO:15) and CDR3 (SEQ ID NO: 18) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 4 shows the alignment of the amino acid sequence of the heavy chainvariable region of 13H5 (SEQ ID NO:19) and 7H9 (SEQ ID NO:21) with thehuman germline VH 1-18 amino acid sequence (SEQ ID NO: 31).

FIG. 5 shows the alignment of the amino acid sequence of the heavy chainvariable region of 13H7 (SEQ ID NO:20) with the human germline VH 4-61amino acid sequence (SEQ ID NO: 32).

FIG. 6 shows the alignment of the amino acid sequence of the light chainvariable region of 13H5 (SEQ ID NO:22), 13H7 (SEQ ID NO:23) and 7H9 (SEQID NO:24) with the human germline VK A27 amino acid sequence (SEQ ID NO:33).

FIG. 7 is a graph showing competition of binding of ¹²⁵I-IFNα 2a toIFNAR-expressing Daudi cells by unlabeled IFNα 2a () versus enhancementof ¹²⁵I-IFNα 2a binding by mAb 13H5 (▾). An isotype control antibody hadno effect on binding (♦).

FIG. 8 is a graph showing binding of ¹²⁵¹-13H5 to Daudi cells in thepresence of IFNα 2a (▪) but not in the absence of IFNα 2a (▴). SpecificIFNα-dependent binding of 13H5 is represented by circles ().

FIG. 9 is a graph showing the results of ADCC assays of Raji cell lysisby fresh human mononuclear cells in the presence of 13H5 (▪), 13H5+IFNα(▴), an isotype control antibody+IFNα (▾), or a positive controlantibody (). Lysis was only seen with the positive control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated monoclonal antibodies thatbind to IFN alpha and that are capable of inhibiting the biologicalactivity of multiple IFN alpha subtypes, but not the biological activityof IFN alpha subtype 21, or IFN beta or IFN omega. The antibodies of theinvention are capable of inhibiting surface expression of cell markersinduced by IFN alpha, inhibiting IP-10 expression induced by IFN alphaand inhibiting dendritic cell development mediated by plasma frompatients with systemic lupus erythematosus (SLE). The invention providesisolated antibodies, methods of making such antibodies, immunoconjugatesand bispecific molecules comprising such antibodies and pharmaceuticalcompositions containing the antibodies, immunconjugates or bispecificmolecules of the invention. The invention also relates to methods ofusing the antibodies to inhibit IFN alpha activity, for example in thetreatment of autoimmune disorders, or for inhibiting or preventingtransplant rejection or in the treatment of graft versus host disease.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The terms “interferon alpha” and “IFN alpha” are used interchangeablyand intended to refer to IFN alpha proteins encoded by a functional geneof the interferon alpha gene locus with 75% or greater sequence identityto IFN alpha 1 (Genbank number NP_(—)076918 or protein encoded byGenbank number NM_(—)024013). Examples of IFN alpha subtypes include IFNalpha 1, alpha 2a, alpha 2b, alpha 4, alpha 5, alpha 6, alpha 7, alpha8, alpha 10, alpha 13, alpha 14, alpha 16, alpha 17 and alpha 21. Theterm “interferon alpha” is intended to encompass recombinant forms ofthe various IFN alpha subtypes, as well as naturally occurringpreparations that comprise IFN alpha proteins, such as leukocyte IFN andlymphoblastoid IFN. The term IFN alpha is not intended to encompass IFNomega alone, although a composition that comprises both IFN alpha andIFN omega is encompassed by the term IFN alpha.

The term “IFN alpha receptor” as used herein is intended to refer tomembers of the IFN alpha receptor family of molecules that are receptorsfor the ligand IFN alpha. Examples of IFN alpha receptors are IFN alphareceptor 1 and IFN alpha receptor 2.

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines, and complement) that results inselective damage to, destruction of, or elimination from the human bodyof invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

A “signal transduction pathway” refers to the biochemical relationshipbetween a variety of signal transduction molecules that play a role inthe transmission of a signal from one portion of a cell to anotherportion of a cell. As used herein, the phrase “cell surface receptor”includes, for example, molecules and complexes of molecules capable ofreceiving a signal and the transmission of such a signal across theplasma membrane of a cell. An example of a “cell surface receptor” ofthe present invention is the IFN alpha receptor 1 or IFN alpha receptor2.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechains thereof. An “antibody” refers to a glycoprotein comprising atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen binding portion thereof. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein asV_(H)) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, C_(H1), C_(H2) and C_(H3). Eachlight chain is comprised of a light chain variable region (abbreviatedherein as V_(L)) and a light chain constant region. The light chainconstant region is comprised of one domain, C_(L). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., IFN alpha). It has been shown that the antigen-binding functionof an antibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H1)domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and C_(H1) domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, V_(L) and V_(H), are coded for by separate genes, they canbe joined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(L) and V_(H)regions pair to form monovalent molecules (known as single chain Fv(scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston etal. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chainantibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds IFN alpha is substantially free of antibodies that specificallybind antigens other than IFN alpha). An isolated antibody thatspecifically binds IFN alpha may, however, have cross-reactivity toother antigens, such as IFN alpha molecules from other species.Moreover, an isolated antibody may be substantially free of othercellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from human germline immunoglobulin sequences.Furthermore, if the antibody contains a constant region, the constantregion also is derived from human germline immunoglobulin sequences. Thehuman antibodies of the invention may include amino acid residues notencoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, the term “human antibody”, as used herein,is not intended to include antibodies in which CDR sequences derivedfrom the germline of another mammalian species, such as a mouse, havebeen grafted onto human framework sequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable regions in which both theframework and CDR regions are derived from human germline immunoglobulinsequences. In one embodiment, the human monoclonal antibodies areproduced by a hybridoma which includes a B cell obtained from atransgenic nonhuman animal, e.g., a transgenic mouse, having a genomecomprising a human heavy chain transgene and a light chain transgenefused to an immortalized cell.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom (described further below), (b)antibodies isolated from a host cell transformed to express the humanantibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by the heavy chain constant region genes.

As used herein, an antibody that “inhibits the biological activity” ofan IFN alpha subtype is intended to refer to an antibody that inhibitsthe activity of that subtype by at least 10%, more preferably at least20%, 30%, 40%, 50%, 60%, 70% or 80%, as compared to the level ofactivity in the absence of the antibody, for example using a functionalassay such as those described in the Examples, such as the Daudi cellproliferation assay. Alternatively, an antibody that “inhibits thebiological activity” of an IFN alpha subtype can refer to an antibodythat inhibits the activity of that subtype with an EC₅₀ of less than 200nM or less, more preferably 100 nM or less, even more preferably 50 nMor less and even more preferably 10 nM or less.

As used herein, an antibody that “does not substantially inhibit thebiological activity” of an IFN alpha subtype, or of IFN beta or IFNomega, is intended to refer to an antibody that inhibits the activity ofthat subtype by at less than 10%, more preferably by less than 5% andeven more preferably by essentially undetectable amounts. Alternatively,an antibody that “does not inhibit the biological activity” of an IFNalpha subtype can refer to an antibody that inhibits the activity ofthat subtype with an EC₅₀ of 300 nM or greater.

As used herein, “specific binding” refers to antibody binding to apredetermined antigen. Typically, the antibody binds with a dissociationconstant (K_(D)) of 10⁻⁸M or less, and binds to the predeterminedantigen with a K_(D) that is at least two-fold less than its K_(D) forbinding to a non-specific antigen (e.g., BSA, casein) other than thepredetermined antigen or a closely-related antigen. The phrases “anantibody recognizing an antigen” and “an antibody specific for anantigen” are used interchangeably herein with the term “an antibodywhich binds specifically to an antigen”.

The term “K_(assoc)” or “K_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “K_(dis)” or “K_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of K_(d) toK_(a) (i.e., K_(d)/K_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. A preferred method for determining the K_(D) ofan antibody is by using surface plasmon resonance, preferably using abiosensor system such as a Biacore® system.

As used herein, the term “high affinity” for an IgG antibody refers toan antibody having a K_(D) of 10⁻⁸ M or less, more preferably 10⁻⁹ M orless and even more preferably 10⁻¹⁰ M or less. However, “high affinity”binding can vary for other antibody isotypes. For example, “highaffinity” binding for an IgM isotype refers to an antibody having a KDof 10⁻⁷ M or less, more preferably 10⁻⁸ M or less.

As used herein, the term “subject” includes any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats,horses, cows chickens, amphibians, reptiles, etc.

Various aspects of the invention are described in further detail in thefollowing subsections.

Anti-IFN Alpha Antibodies

The antibodies of the invention are characterized by particularfunctional features or properties of the antibodies. For example, inparticular embodiments, the antibodies bind specifically to multiplesubtypes of IFN alpha, such as IFN alpha 2a and IFN alpha 2b.Preferably, an antibody of the invention binds to IFN alpha 2a and/oralpha 2b with high affinity, for example with a K_(D) of 10⁻⁸ M or lessor 10⁻⁹ M or less or even 10⁻¹⁰ M or less. In a preferred embodiment,the antibody binds to human IFN alpha 2a and human IFN alpha 2b. Thebinding affinity and kinetics of the antibodies of the invention can beexamined by, for example, Biacore analysis as described in the Examples.

Furthermore, in other embodiments, the antibodies of the inventionexhibit various functional properties. For example, the antibodies maybe capable of inhibiting the biological activity of multiple IFN alphasubtypes but may not substantially inhibit the biological activity ofIFN alpha 21. The antibodies also may not substantially inhibit thebiological activity of IFN beta or IFN omega. The antibodies of theinvention also may be capable of inhibiting IFN-induced surfaceexpression of cell markers, such as CD38 or MHC Class I, on normal humanperipheral blood mononuclear cells. The antibodies also may be capableof inhibiting IFN-induced expression of IP-10 by normal human peripheralblood mononuclear cells. Inhibition of biological activity of IFN alphasubtypes, IFN beta and/or IFN omega can be evaluated using functionalassays such as those described in the Examples, such as a Daudi cellproliferation assay.

Still further, the antibodies may be capable of inhibiting dendriticcell development mediated by plasma of patients with systemic lupuserythematosus (SLE). Dendritic cell development can be assessed byexamining the expression of cell surface markers, such as CD38, MHCClass I and/or CD123, as described in the Examples.

In certain preferred embodiments, an antibody of the invention inhibitsthe biological activity of IFN alpha by a non-competitive mechanism ofaction, i.e., the antibody does not compete for binding of IFN alpha toIFNAR. Rather, such an antibody becomes associated with cell-surfaceIFNAR in the presence of IFN alpha and inhibits cell signaling throughIFNAR. In other preferred embodiments, an antibody having these bindingproperties does not exhibit significant ADCC activity. Assays forexamining these functional properties of the antibody are known in theart, such as the assays described in Examples 8 and 9. For example, theability of the antibody to inhibit binding of radiolabeled IFN alpha toIFNAR-expressing cells can be examined. The inability of the antibody toinhibit the binding of radiolabeled IFN alpha to IFNAR is indicative ofa non-competitive mechanism of action. To further examine this mechanismof action, the binding of radiolabeled antibody, in the presence orabsence of IFN alpha, to IFNAR-expressing cells can be assayed. Bindingof the radiolabeled antibody to IFNAR-expressing cells in the presence,but not the absence, of IFN alpha is indicative this mechanism ofaction.

In a preferred embodiment, antibodies of the invention bind to the IFNalpha—IFNAR complex with a greater affinity (e.g., K_(D)) than to IFNalpha alone (one or more subtypes) and/or to IFNAR alone. For example,in certain embodiments, antibodies of the invention bind the IFNalpha-IFNAR complex with a K_(D) of 10⁻⁸ M or greater affinity, a K_(D)of 10⁻⁹ M or greater affinity, or a K_(D) of 10¹⁰ M or greater affinity.

In another preferred embodiment, antibodies of the invention arebispecific for IFN alpha (one or more subtypes) and IFNAR (IFNAR1 and/orIFNAR2), meaning that the antibodies associate with both IFN alpha andIFNAR (IFNAR1 and/or IFNAR2). Accordingly, the present inventionincludes bispecific molecules comprising at least one first bindingspecificity for IFN alpha and a second binding specificity for IFNAR1,wherein, for example, the second binding specificity for IFNAR1 can beformed by the association of the antibody with IFN alpha. The presentinvention also includes bispecific molecules comprising at least onebinding specificity for IFN alpha and a second binding specificity forIFNAR2, wherein, for example, the second binding specificity for INFAR2can be formed by association of the antibody with IFN alpha.

Monoclonal Antibodies 13H5, 13H7 and 7H9

Preferred antibodies of the invention are the human monoclonalantibodies 13H5, 13H7, and 7H9, isolated and structurally characterizedas described in the Examples. The V_(H) amino acid sequences of 13H5,13H7, and 7H9 are shown in SEQ ID NOs: 19, 20, and 21, respectively. TheV_(L) amino acid sequences of 13H5, 13H7, and 7H9 are shown in SEQ IDNOs: 22, 23 and 24, respectively. Given that each of these antibodiescan bind to IFN alpha, the V_(H) and V_(L) sequences can be “mixed andmatched” to create other anti-IFN alpha binding molecules of theinvention. IFN alpha binding or neutralizing activity of such “mixed andmatched” antibodies can be tested using the binding assays describedabove and in the Examples (e.g., ELISA, Biacore analysis, Daudi cellproliferation assay). Preferably, the V_(H) sequences of 13H5 and 7H9are mixed and matched, since these antibodies use V_(H) sequencesderived from the same germline sequence (VH 1-18) and thus they exhibitstructural similarity. Additionally or alternatively, the V_(L)sequences of 13H5, 13H7 and 7H9 can be mixed and matched, since theseantibodies use V_(L) sequences derived from the same germline sequence(V_(k) A27) and thus they exhibit structural similarity.

Accordingly, in one aspect, the invention provides an isolatedmonoclonal antibody, or antigen binding portion thereof, comprising:

(a) a heavy chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 19, 20, and 21; and

(b) a light chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 22, 23, and 24;

wherein the antibody inhibits the biological activity of interferonalpha.

Preferred heavy and light chain combinations include:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 19; and (b) a light chain variable region comprising theamino acid sequence of SEQ ID NO:22; or

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 20; and (b) a light chain variable region comprising theamino acid sequence of SEQ ID NO:23; or

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 21; and (b) a light chain variable region comprising theamino acid sequence of SEQ ID NO:24.

In another aspect, the invention provides antibodies that comprise theheavy chain and light chain CDR1s, CDR2s, and CDR3s of 13H5, 13H7, and7H9, or combinations thereof. The amino acid sequences of the V_(H)CDR1s of 13H5, 13H7, and 7H9 are shown in SEQ IN NOs: 1, 2, and 3. Theamino acid sequences of the V_(H) CDR2s of 13H5, 13H7, and 7H9 are shownin SEQ IN NOs: 4, 5, and 6. The amino acid sequences of the V_(H) CDR3sof 13H5, 13H7, and 7H9 are shown in SEQ IN NOs: 7, 8, and 9. The aminoacid sequences of the V_(L) CDR1s of 13H5, 13H7, and 7H9 are shown inSEQ IN NOs: 10, 11, and 12. The amino acid sequences of the V_(L) CDR2sof 13H5, 13H7, and 7H9 are shown in SEQ IN NOs: 13, 14, and 15. Theamino acid sequences of the V_(L) CDR3s of 13H5, 13H7, and 7H9 are shownin SEQ IN NOs: 16, 17, and 18. The CDR regions are delineated using theKabat system (Kabat. E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242).

Given that each of these antibodies was selected based on IFN bindingactivity and that antigen-binding specificity is provided primarily bythe CDR1, 2 and 3 regions, the V_(H) CDR1, 2 and 3 sequences and V_(L)CDR1, 2 and 3 sequences can be “mixed and matched” (i.e., CDRs fromdifferent antibodies can be mixed and match, although each antibody mustcontain a V_(H) CDR1, 2 and 3 and a V_(L) CDR1, 2 and 3) to create otheranti-IFN alpha molecules of the invention. IFN alpha binding of such“mixed and matched” antibodies can be tested using the binding assaysdescribed in the Examples (e.g., ELISA and/or Biacore). Preferably, whenV_(H) CDR sequences are mixed and matched, the CDR1, CDR2 and/or CDR3sequence from a particular V_(H) sequence is replaced with astructurally similar CDR sequence(s). Likewise, when V_(L) CDR sequencesare mixed and matched, the CDR1, CDR2 and/or CDR3 sequence from aparticular V_(L) sequence preferably is replaced with a structurallysimilar CDR sequence(s). For example, the V_(H) CDR1s of 13H5 and 7H9share some structural similarity and therefore are amenable to mixingand matching. It will be readily apparent to the ordinarily skilledartisan that novel V_(H) and V_(L) sequences can be created bysubstituting one or more V_(H) and/orV_(L CDR region sequences with structurally similar sequences from the CDR sequences disclosed herein for monoclonal antibodies)13H5, 13H7 and 7H9.

Accordingly, in another aspect, the invention provides an isolatedmonoclonal antibody, or antigen binding portion thereof comprising:

(a) a heavy chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1, 2, and 3;

(b) a heavy chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 4, 5, and 6;

(c) a heavy chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 7, 8, and 9;

(d) a light chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 10, 11, and 12;

(e) a light chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 13, 14, and 15; and

(f) a light chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 16, 17, and 18;

wherein the antibody the antibody inhibits the biological activity ofinterferon alpha.

In a preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 1;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 4;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 7;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 10;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 13; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 16.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 2;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 5;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 8;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 11;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 14; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 17.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 3;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 6;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 9;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 12;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 15; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 18.

Antibodies Having Particular Germline Sequences

In certain embodiments, an antibody of the invention comprises a heavychain variable region from a particular germline heavy chainimmunoglobulin gene and/or a light chain variable region from aparticular germline light chain immunoglobulin gene.

For example, in a preferred embodiment, the invention provides anisolated monoclonal antibody, or an antigen-binding portion thereof,therein the antibody:

(a) comprises a heavy chain variable region of a human VH 1-18 or 4-61gene;

(b) comprises a light chain variable region of a human Vk A27 gene; and

(c) the antibody inhibits the biological activity of interferon alpha.

In one embodiment, the antibody comprises a heavy chain variable regionof a human VH 1-18 gene. Examples of antibodies having a VH and Vk genesequence of VH 1-18 and Vk A27, respectively, include 13H5 and 7H9. Inanother embodiment, the antibody comprises a heavy chain variable regionof a human VH 4-61 gene. An example of an antibody having a VH and Vkgene sequence of VH 4-61 and Vk A27, respectively, is 13H7.

As used herein, a human antibody comprises heavy or light chain variableregions “of” (i.e., the products of) or “derived from” a particulargermline sequence if the variable regions of the antibody are obtainedfrom a system that uses human germline immunoglobulin genes. Suchsystems include immunizing a transgenic mouse carrying humanimmunoglobulin genes with the antigen of interest or screening a humanimmunoglobulin gene library displayed on phage with the antigen ofinterest. A human antibody that is “of” (i.e., the product of) or“derived from” a human germline immunoglobulin sequence can beidentified as such by comparing the amino acid sequence of the humanantibody to the amino acid sequences of human germline immunoglobulinsand selecting the human germline immunoglobulin sequence that is closestin sequence (i.e., greatest % identity) to the sequence of the humanantibody. A human antibody that is “of” (i.e., the product of) or“derived from” a particular human germline immunoglobulin sequence maycontain amino acid differences as compared to the germline sequence, dueto, for example, naturally-occurring somatic mutations or intentionalintroduction of site-directed mutation. However, a selected humanantibody typically is at least 90% identical in amino acids sequence toan amino acid sequence encoded by a human germline immunoglobulin geneand contains amino acid residues that identify the human antibody asbeing human when compared to the germline immunoglobulin amino acidsequences of other species (e.g., murine germline sequences). In certaincases, a human antibody may be at least 95%, or even at least 96%, 97%,98%, or 99% identical in amino acid sequence to the amino acid sequenceencoded by the germline immunoglobulin gene. Typically, a human antibodyderived from a particular human germline sequence will display no morethan 10 amino acid differences from the amino acid sequence encoded bythe human germline immunoglobulin gene. In certain cases, the humanantibody may display no more than 5, or even no more than 4, 3, 2, or 1amino acid difference from the amino acid sequence encoded by thegermline immunoglobulin gene.

Homologous Antibodies

In yet another embodiment, an antibody of the invention comprises heavyand light chain variable regions comprising amino acid sequences thatare homologous to the amino acid sequences of the preferred antibodiesdescribed herein, and wherein the antibodies retain the desiredfunctional properties of the anti-IFN alpha antibodies of the invention.

For example, the invention provides an isolated monoclonal antibody, orantigen binding portion thereof, comprising a heavy chain variableregion and a light chain variable region, wherein:

(a) the heavy chain variable region comprises an amino acid sequencethat is at least 80% homologous to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 19, 20, and 21;

(b) the light chain variable region comprises an amino acid sequencethat is at least 80% homologous to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 22, 23, and 24;

(c) the antibody inhibits the biological activity of multiple IFN alphasubtypes but does not substantially inhibit the biological activity ofIFN alpha 21;

(d) the antibody exhibits at least one of the following properties:

-   -   (i) the antibody does not substantially inhibit the biological        activity of IFN beta or IFN omega;    -   (ii) the antibody inhibits IFN-induced surface expression of        CD38 or MHC Class I on peripheral blood mononuclear cells;    -   (iii) the antibody inhibits IFN-induced expression of IP-10 by        peripheral blood mononuclear cells;    -   (iv) the antibody inhibits dendritic cell development mediated        by systemic lupus erythematosus (SLE) plasma.

In other embodiments, the V_(H) and/or V_(L) amino acid sequences may be85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequences setforth above. An antibody having V_(H) and V_(L) regions having high(i.e., 80% or greater) homology to the V_(H) and V_(L) regions of SEQ IDNOs: 19, 20, and 21 and 22, 23, and 24, respectively, can be obtained bymutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleicacid molecules encoding SEQ ID NOs: 19, 20, and 21 and/or 22, 23, and24, followed by testing of the encoded altered antibody for retainedfunction (i.e., the functions set forth in (c) and (d) above) using thefunctional assays described herein.

As used herein, the percent homology between two amino acid sequences isequivalent to the percent identity between the two sequences. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17 (1988)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the presentinvention can further be used as a “query sequence” to perform a searchagainst public databases to, for example, identify related sequences.Such searches can be performed using the XBLAST program (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to the antibody molecules of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (1997) NucleicAcids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

Antibodies with Conservative Modifications

In certain embodiments, an antibody of the invention comprises a heavychain variable region comprising CDR1, CDR2 and CDR3 sequences and alight chain variable region comprising CDR1, CDR2 and CDR3 sequences,wherein one or more of these CDR sequences comprise specified amino acidsequences based on the preferred antibodies described herein (e.g.,13H5, 13H7, or 7H9), or conservative modifications thereof, and whereinthe antibodies retain the desired functional properties of the anti-IFNalpha antibodies of the invention. For example, preferred antibodies ofthe invention include those in which the heavy chain variable regionCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 3, orconservative modifications thereof, and the light chain variable regionCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 6, orconservative modifications thereof. Accordingly, the invention providesan isolated monoclonal antibody, or antigen binding portion thereof,comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3sequences and a light chain variable region comprising CDR1, CDR2, andCDR3 sequences, wherein:

(a) the heavy chain variable region CDR3 sequence comprises the aminoacid sequence selected from the group consisting of SEQ ID NO: 7, 8, and9, and conservative modifications thereof;

(b) the light chain variable region CDR3 sequence comprises the aminoacid sequence selected from the group consisting of SEQ ID NO: 16, 17,and 18, and conservative modifications thereof;

(c) the antibody inhibits the biological activity of multiple IFN alphasubtypes but does not substantially inhibit the biological activity ofIFN alpha 21;

(d) the antibody exhibits at least one of the following properties:

-   -   (i) the antibody does not substantially inhibit the biological        activity of IFN beta or IFN omega;    -   (ii) the antibody inhibits IFN-induced surface expression of        CD38 or MHC Class I on peripheral blood mononuclear cells;    -   (iii) the antibody inhibits IFN-induced expression of IP-10 by        peripheral blood mononuclear cells;    -   (iv) the antibody inhibits dendritic cell development mediated        by systemic lupus erythematosus (SLE) plasma.

In a further embodiment, the heavy chain variable region CDR2 sequencecomprises the amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NO: 4, 5, and 6, and conservativemodifications thereof; and the light chain variable region CDR2 sequencecomprises the amino acid sequence selected from the group consisting ofamino acid sequences SEQ ID NO: 13, 14, and 15, and conservativemodifications thereof. In a still further embodiment, the heavy chainvariable region CDR1 sequence comprises the amino acid sequence selectedfrom the group consisting of amino acid sequences of SEQ ID NO: 1, 2,and 3, and conservative modifications thereof; and the light chainvariable region CDR1 sequence comprises the amino acid sequence selectedfrom the group consisting of amino acid sequences of SEQ ID NO: 10, 11,and 12, and conservative modifications thereof.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of the invention by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions are ones in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, praline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, oneor more amino acid residues within the CDR regions of an antibody of theinvention can be replaced with other amino acid residues from the sameside chain family and the altered antibody can be tested for retainedfunction (i.e., the functions set forth in (c) and (d) above) using thefunctional assays described herein.

Antibodies that Bind to the Same Epitope as Anti-IFN Alpha Antibodies ofthe Invention

In another embodiment, the invention provides antibodies that bind tothe same epitope as do the various human IFN alpha antibodies of theinvention provided herein, such as other human antibodies that bind tothe same epitope as the 13H5, 13H7, and 7H9 antibodies described herein.Such antibodies can be identified based on their ability tocross-compete (e.g., to competitively inhibit the binding of, in astatistically significant manner) with other antibodies of theinvention, such as 13H5, 13H7 or 7H9, in standard IFN alpha bindingassays. For example, as demonstrated in the Examples by Biacoreanalysis, 13H5 binds with high affinity to IFN alpha 2a and IFN alpha2b. Accordingly, in one embodiment, the invention provides antibodies,preferably human antibodies, that compete for binding to IFN alpha 2a orIFN alpha 2b with another antibody of the invention (e.g., 13H5, 13H7 or7H9). The ability of a test antibody to inhibit the binding of, e.g.,13H5, 13H7 or 7H9 to IFN alpha 2a or IFN alpha 2b demonstrates that thetest antibody can compete with that antibody for binding to IFN alpha 2aor IFN alpha 2b; such an antibody may, according to non-limiting theory,bind to the same or a related (e.g., a structurally similar or spatiallyproximal) epitope on IFN alpha 2a or IFN alpha 2b as the antibody withwhich it competes. In a preferred embodiment, the antibody that binds tothe same epitope on IFN alpha 2a or IFN alpha 2b as, e.g., 13H5, 13H7,or 7H9, is a human monoclonal antibody. Such human monoclonal antibodiescan be prepared and isolated as described in the Examples.

Engineered and Modified Antibodies

An antibody of the invention further can be prepared using an antibodyhaving one or more of the V_(H) and/or V_(L) sequences disclosed hereinas starting material to engineer a modified antibody, which modifiedantibody may have altered properties from the starting antibody. Anantibody can be engineered by modifying one or more residues within oneor both variable regions (i.e., V_(H) and/or V_(L)), for example withinone or more CDR regions and/or within one or more framework regions.Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example to alterthe effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al. (1998) Nature332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. etal. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.)

Accordingly, another embodiment of the invention pertains to an isolatedmonoclonal antibody, or antigen binding portion thereof, comprising aheavy chain variable region comprising CDR1, CDR2, and CDR3 sequencescomprising the amino acid sequences selected from the group consistingof SEQ ID NO: 1, 2, and 3, SEQ ID NO: 4, 5, and 6 and SEQ ID NO: 7, 8,and 9, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 sequences comprising the amino acid sequences selectedfrom the group consisting of SEQ ID NO:10, 11, and 12, SEQ ID NO: 13,14, and 15 and SEQ ID NO: 16, 17, and 18, respectively. Thus, suchantibodies contain the V_(H) and V_(L) CDR sequences of monoclonalantibodies 13H5, 13H7 or 7H9, yet may contain different frameworksequences from these antibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), aswell as in Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al.(1992) “The Repertoire of Human Germline V_(H) Sequences Reveals aboutFifty Groups of V_(H) Segments with Different Hypervariable Loops” J.Mol. Biol. 227:776-798; and Cox, J. P. L. et al. (1994) “A Directory ofHuman Germ-line V_(H) Segments Reveals a Strong Bias in their Usage”Eur. J. Immunol. 24:827-836; the contents of each of which are expresslyincorporated herein by reference.

Preferred framework sequences for use in the antibodies of the inventionare those that are structurally similar to the framework sequences usedby selected antibodies of the invention, e.g., similar to the VH 1-18 or4-61 and VK A27 framework sequences used by the preferred monoclonalantibodies of the invention. The V_(H) CDR1, 2 and 3 sequences, and theV_(L) CDR1, 2 and 3 sequences can be grafted onto framework regions thathave the same sequence as that found in the germline immunoglobulin genefrom which the framework sequence derive, or the CDR sequences can begrafted onto framework regions that contain one or more mutations ascompared to the germline sequences. For example, it has been found thatin certain instances it is beneficial to mutate residues within theframework regions to maintain or enhance the antigen binding ability ofthe antibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al).

Another type of variable region modification is to mutate amino acidresidues within the V_(H) and/or V_(L) CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest. Site-directed mutagenesis or PCR-mediatedmutagenesis can be performed to introduce the mutation(s) and the effecton antibody binding, or other functional property of interest, can beevaluated in in vitro or in vivo assays as described herein and providedin the Examples. Preferably conservative modifications (as discussedabove) are introduced. The mutations may be amino acid substitutions,additions or deletions, but are preferably substitutions. Moreover,typically no more than one, two, three, four or five residues arealtered within a CDR region are altered.

Accordingly, in another embodiment, the invention provides isolatedanti-IFN alpha monoclonal antibodies, or antigen binding portionsthereof, comprising a heavy chain variable region comprising: (a) aV_(H) CDR1 region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1, 2, and 3, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to SEQ ID NOs: 1, 2, or 3; (b) a V_(H) CDR2region comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4, 5, and 6, or an amino acid sequence havingone, two, three, four or five amino acid substitutions, deletions oradditions as compared to SEQ ID NOs: 4, 5, or 6; (c) a V_(H) CDR3 regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 7, 8, and 9, or an amino acid sequence having one, two,three, four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NOs: 7, 8, or 9; (d) a V_(L) CDR1 region comprisingan amino acid sequence selected from the group consisting of SEQ ID NOs:10, 11, and 12, or an amino acid sequence having one, two, three, fouror five amino acid substitutions, deletions or additions as compared toSEQ ID NOs: 10, 11, or 12; (e) a V_(L) CDR2 region comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 13, 14,and 15, or an amino acid sequence having one, two, three, four or fiveamino acid substitutions, deletions or additions as compared to SEQ IDNOs: 13, 14, or 15; and (f) a V_(L) CDR3 region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 16, 17, and18, or an amino acid sequence having one, two, three, four or five aminoacid substitutions, deletions or additions as compared to SEQ ID NOs:16, 17, or 18.

Engineered antibodies of the invention include those in whichmodifications have been made to framework residues within V_(H) and/orV_(L), e.g. to improve the properties of the antibody. Typically suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation maycontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived. For example, for 13H5, amino acidresidue #81 (within FR3) of V_(H) is a leucine whereas this residue inthe corresponding VH 1-18 germline sequence is a methionine (see FIG.4). To return the framework region sequences to their germlineconfiguration, the somatic mutations can be “backmutated” to thegermline sequence by, for example, site-directed mutagenesis orPCR-mediated mutagenesis (e.g., residue 81 of the V_(H) of 13H5 can be“backmutated” from leucine to methionine). Such “backmutated” antibodiesare also intended to be encompassed by the invention.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in father detail in U.S. PatentPublication No. 20030153043 by Can et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the invention may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter it's glycosylation, again toalter one or more functional properties of the antibody. Each of theseembodiments is described in further detail below. The numbering ofresidues in the Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fe hinge region of an antibody is mutated todecrease the biological half life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase itsbiological half life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the antibody. For example, one or more aminoacids selected from amino acid residues 234, 235, 236, 237, 297, 318,320 and 322 can be replaced with a different amino acid residue suchthat the antibody has an altered affinity for an effector ligand butretains the antigen-binding ability of the parent antibody. The effectorligand to which affinity is altered can be, for example, an Fc receptoror the C1 component of complement. This approach is described in furtherdetail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered C1q binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551 by Idusogie etal.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351 by Bodmer et al.

In yet another example, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids at the followingpositions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268,269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326,327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378,382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. Thisapproach is described further in PCT Publication WO 00/42072 by Presta.Moreover, the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII andFeRn have been mapped and variants with improved binding have beendescribed (see Shields, R. L. et al. (2001) J. Biol. Chem.276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334and 339 were shown to improve binding to FcγRIII. Additionally, thefollowing combination mutants were shown to improve FcγRIII binding:T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333AfK334A.

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of the invention to thereby produce an antibodywith altered glycosylation. For example, EP 1,176,195 by Hanai et al.describes a cell line with a functionally disrupted FUT8 gene, whichencodes a fucosyl transferase, such that antibodies expressed in such acell line exhibit hypofucosylation. PCT Publication WO 03/035835 byPresta describes a variant CHO cell line, Lec13 cells, with reducedability to attach fucose to Asn(297)-linked carbohydrates, alsoresulting in hypofucosylation of antibodies expressed in that host cell(see also Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740).PCT Publication WO 99/54342 by Umana et al. describes cell linesengineered to express glycoprotein-modifying glycosyl transferases(e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).

Another modification of the antibodies herein that is contemplated bythe invention is pegylation. An antibody can be pegylated to, forexample, increase the biological (e.g., serum) half life of theantibody. To pegylate an antibody, the antibody, or fragment thereof,typically is reacted with polyethylene glycol (PEG), such as a reactiveester or aldehyde derivative of PEG, under conditions in which one ormore PEG groups become attached to the antibody or antibody fragment.Preferably, the pegylation is carried out via an acylation reaction oran alkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies of the invention. See for example, EP 0 154 316 byNishimura et al. and EP 0 401 384 by Ishikawa et al.

Modified Antibodies with Increased Stability

In another aspect, the invention provides modified forms of the 13H5antibody that exhibit increased stability as compared to wild-type 13H5.As described in further detail in Example 10, the 13H5 antibody containsa deamidation site at Asn-55 within CDR2 of the V_(H) chain. The aminoacid sequence at this site, from positions 55 to 58) is N G N T (aminoacid residues 55-58 of SEQ ID NO: 19). Accordingly, in certainembodiments, the amino acid sequence of the 13H5 V_(H) chain is mutatedat position 55 from asparagine to a different amino acid. Additionallyor alternatively, amino acid positions around Asn-55 that influencedeamidation can be mutated. Preferred amino acid substitutions atposition 55 include aspartic acid and glutamine, with glutamine beingmore preferred. The amino acid sequence of 13H5 with a N55D substitutionis shown in SEQ ID NO: 34. The amino acid sequence of 13H5 with a N55Qsubstitution is shown in SEQ ID NO: 35. In another embodiment, Asn-57 ofthe 13H5 V_(H) chain is also mutated, together with mutation of Asn-55.A preferred amino acid substitution at position 57 is glutamine. Theamino acid sequence of 13H5 with N55Q and N57Q substitutions is shown inSEQ ID NO: 36. These three mutated antibodies exhibit increasedstability, under forced deamidation conditions, as compared to wild-type13H5, as described further in Example 11.

In another embodiment, the glycine at amino acid position 56 is mutatedto an alanine (G56A), since it has been determined from model peptidesthat the rate of deamidation is approximately 20-fold less with analanine adjacent to the asparagine, rather than a glycine adjacent tothe alanine (see e.g., Ahern, T. and Manning, M. C., eds. Stability ofProtein Pharmaceuticals, Pharmaceutical Biotechnology, volume 2, chapter1, pages 1-30). Thus, the G56A mutation represents a balance betweendecreased reactivity and minimal structural change to the wild typesequence, thus increasing stability while maintaining activity. Theamino acid sequence of 13H5 with a G56A substitution is shown in SEQ IDNO: 37.

Accordingly, in various embodiments, the invention provides an IFN alphaantibody of the invention having an amino acid substitution at Asn-55,Gly-56 and/or Asn-57 of the CDR2 of the 13H5 V_(H) chain, the wild typesequence of which is shown set forth in SEQ ID NO: 19. Preferred mutatedantibodies comprise a heavy chain variable region comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 34, 35,36 and 37. Preferably, the antibody VH chain is paired with the VK chainof 13H5, as set forth in SEQ ID NO: 22.

Methods of Engineering Antibodies

As discussed above, the anti-IFN alpha antibodies having VH and VLsequences disclosed herein can be used to create new anti-IFN alphaantibodies by modifying the VH and/or VL sequences, or the constantregion(s) attached thereto. Thus, in another aspect of the invention,the structural features of an anti-IFN alpha antibody of the invention,e.g. 13H5, 13H7 or 7H9, are used to create structurally related anti-IFNalpha antibodies that retain at least one functional property of theantibodies of the invention, such as binding to IFN alpha. For example,one or more CDR regions of 13H5, 13H7 or 7H9, or mutations thereof, canbe combined recombinantly with known framework regions and/or other CDRsto create additional, recombinantly-engineered, anti-IFN alphaantibodies of the invention, as discussed above. Other types ofmodifications include those described in the previous section. Thestarting material for the engineering method is one or more of the V_(H)and/or V_(L) sequences provided herein, or one or more CDR regionsthereof. To create the engineered antibody, it is not necessary toactually prepare (i.e., express as a protein) an antibody having one ormore of the V_(H) and/or V_(L) sequences provided herein, or one or moreCDR regions thereof. Rather, the information contained in thesequence(s) is used as the starting material to create a “secondgeneration” sequence(s) derived from the original sequence(s) and thenthe “second generation” sequence(s) is prepared and expressed as aprotein.

Accordingly, in another embodiment, the invention provides a method forpreparing an anti-IFN alpha antibody comprising:

(a) providing: (i) a heavy chain variable region antibody sequencecomprising a CDR1 amino acid sequence selected from the group consistingof SEQ ID NOs: 1, 2 and 3; and/or a CDR2 amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 4, 5, and 6; and/or a CDR3amino acid sequence selected from the group consisting of SEQ ID NOs: 7,8, and 9; and/or (ii) a light chain variable region antibody sequencecomprising a CDR1 amino acid sequence selected from the group consistingof SEQ ID NOs: 10, 11, and 12; and/or a CDR2 amino acid sequenceselected from the group consisting of SEQ ID NOs: 13, 14, and 15; and/ora CDR3 amino acid sequence selected from the group consisting of SEQ IDNOs: 13, 14, and 15;

(b) altering at least one amino acid residue within the heavy chainvariable region antibody sequence and/or the light chain variable regionantibody sequence to create at least one altered antibody sequence; and

(c) expressing the altered antibody sequence as a protein.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence.

Preferably, the antibody encoded by the altered antibody sequence(s) isone that retains one, some or all of the functional properties of theanti-IFN alpha antibodies described herein, which functional propertiesinclude, but are not limited to:

-   -   (i) inhibiting the biological activity of interferon alpha;    -   (ii) inhibiting the biological activity of multiple IFN alpha        subtypes but not substantially inhibiting the biological        activity of IFN alpha 21;    -   (iii) not substantially inhibiting the biological activity of        IFN beta or IFN omega;    -   (iv) inhibiting IFN-induced surface expression of CD38 or MHC        Class I on peripheral blood mononuclear cells;    -   (v) inhibiting IFN-induced expression of IP-10 by peripheral        blood mononuclear cells;    -   (vi) inhibiting dendritic cell development mediated by systemic        lupus erythematosus (SLE) plasma;    -   (vii) binding to human interferon alpha 2a with high affinity;    -   (viii) binding to human interferon alpha 2b with high affinity.

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein. Forexample, the ability of the antibody to bind IFN alpha can be determinedusing standard binding assays, such as those set forth in the Examples(e.g., ELISAs and/or Biacores). The ability of the antibody to inhibitvarious functional activities of interferon alpha can be determinedusing assays such as those described in the Examples (e.g., Daudi cellproliferation, IFN-induced cell marker expression, IFN-induced IP-10expression etc.)

In certain embodiments of the methods of engineering antibodies of theinvention, mutations can be introduced randomly or selectively along allor part of an anti-IFN alpha antibody coding sequence (e.g., 13H5 codingsequence) and the resulting modified anti-IFN alpha antibodies can bescreened for binding activity and/or other functional properties asdescribed herein. Mutational methods have been described in the art. Forexample, PCT Publication WO 02/092780 by Short describes methods forcreating and screening antibody mutations using saturation mutagenesis,synthetic ligation assembly, or a combination thereof. Alternatively,PCT Publication WO 03/074679 by Lazar et al., describes methods of usingcomputational screening methods to optimize physiochemical properties ofantibodies.

Nucleic Acid Molecules Encoding Antibodies of the Invention

Another aspect of the invention pertains to nucleic acid molecules thatencode the antibodies of the invention. The nucleic acids may be presentin whole cells, in a cell lysate, or in a partially purified orsubstantially pure form. A nucleic acid is “isolated” or “renderedsubstantially pure” when purified away from other cellular components orother contaminants, e.g., other cellular nucleic acids or proteins, bystandard techniques, including alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art. See, F. Ausubel, et al., ed. (1987) Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York. Anucleic acid of the invention can be, for example, DNA or RNA and may ormay not contain intronic sequences. In a preferred embodiment, thenucleic acid is a cDNA molecule.

Nucleic acids of the invention can be obtained using standard molecularbiology techniques. For antibodies expressed by hybridomas (e.g.,hybridomas prepared from transgenic mice carrying human immunoglobulingenes as described further below), cDNAs encoding the light and heavychains of the antibody made by the hybridoma can be obtained by standardPCR amplification or cDNA cloning techniques. For antibodies obtainedfrom an immunoglobulin gene library (e.g., using phage displaytechniques), nucleic acid encoding the antibody can be recovered fromthe library.

Preferred nucleic acids molecules of the invention are those encodingthe V_(H) and V_(L) sequences of the 13H5, 13H7, or 7H9 monoclonalantibodies. DNA sequences encoding the V_(H) sequences of 13H5, 13H7,and 7H9 are shown in SEQ ID NOs: 25, 26, and 27, respectively. DNAsequences encoding the V_(L) sequences of 13H5, 13H7, and 7H9 are shownin SEQ ID NOs: 28, 29, and 30, respectively.

Once DNA fragments encoding V_(H) and V_(L) segments are obtained, theseDNA fragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to a scFvgene. In these manipulations, a V_(L)- or V_(H)-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker. The term“operatively linked”, as used in this context, is intended to mean thatthe two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions (CH1,CH2 and CH3). The sequences of human heavy chain constant region genesare known in the art (see e.g., Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene,the V_(H)-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, CK. The sequences of humanlight chain constant region genes are known in the art (see e.g., Kabat,E. A., et al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242) and DNA fragments encompassing these regionscan be obtained by standard PCR amplification. The light chain constantregion can be a kappa or lambda constant region, but most preferably isa kappa constant region.

To create a scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the V_(H) and V₁sequences can be expressed as a contiguous single-chain protein, withthe V_(L) and V_(H) regions joined by the flexible linker (see e.g.,Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature348:552-554).

Production of Monoclonal Antibodies of the Invention

Monoclonal antibodies (mAbs) of the present invention can be produced bya variety of techniques, including conventional monoclonal antibodymethodology e.g., the standard somatic cell hybridization technique ofKohler and Milstein (1975) Nature 256: 495. Although somatic cellhybridization procedures are preferred, in principle, other techniquesfor producing monoclonal antibody can be employed e.g., viral oroncogenic transformation of B lymphocytes.

The preferred animal system for preparing hybridomas is the murinesystem. Hybridoma production in the mouse is a very well-establishedprocedure. Immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are known in the art. Fusion partners(e.g., murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present invention can beprepared based on the sequence of a murine monoclonal antibody preparedas described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the murine hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, the murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,the murine CDR regions can be inserted into a human framework usingmethods known in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter,and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 toQueen et al.).

In a preferred embodiment, the antibodies of the invention are humanmonoclonal antibodies. Such human monoclonal antibodies directed againstIFN alpha can be generated using transgenic or transchromosomic micecarrying parts of the human immune system rather than the mouse system.These transgenic and transchromosomic mice include mice referred toherein as HuMAb mice and KM mice, respectively, and are collectivelyreferred to herein as “human Ig mice.”

The HuMAb Mouse® (Medarex, Inc.) contains human immunoglobulin geneminiloci that encode unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al.(1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N.(1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. andLonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546). The preparationand use of HuMab mice, and the genomic modifications carried by suchmice, is further described in Taylor, L. et al. (1992) Nucleic AcidsResearch 20:6287-6295; Chen, J. et al. (1993) International Immunology5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sei. USA90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. etal. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol.152:2912-2920; Taylor, L. et al. (1994) International Immunology 6:579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851, the contents of all of which are hereby specificallyincorporated by reference in their entirety. See further, U.S. Pat. Nos.5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397;5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay;U.S. Pat. No. 5,545,807 to Surani et al.; PCT Publication Nos. WO92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO99/45962, all to Lonberg and Kay; and PCT Publication No. WO 01/14424 toKorman et al.

In another embodiment, human antibodies of the invention can be raisedusing a mouse that carries human immunoglobulin sequences on transgenesand transchomosomes, such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto herein as “KM mice”, are described in detail in PCT Publication WO02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-IFN alpha antibodies of the invention. For example, an alternativetransgenic system referred to as the Xenomouse (Abgenix, Inc.) can beused; such mice are described in, for example, U.S. Pat. Nos. 5,939,598;6,075,181; 6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-IFN alpha antibodies of the invention. For example, mice carryingboth a human heavy chain transchromosome and a human light chaintranchromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al. (2002)Nature Biotechnology 20:889-894) and can be used to raise anti-IFN alphaantibodies of the invention.

Human monoclonal antibodies of the invention can also be prepared usingphage display methods for screening libraries of human immunoglobulingenes. Such phage display methods for isolating human antibodies areestablished in the art. See for example: U.S. Pat. Nos. 5,223,409;5,403,484; and 5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 toMcCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731;6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

Human monoclonal antibodies of the invention can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

Immunization of Human Ig Mice

When human Ig mice are used to raise human antibodies of the invention,such mice can be immunized with a purified or recombinant preparation ofIFN alpha antigen, as described by Lonberg, N. et al. (1994) Nature368(6474): 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851; and PCT Publication WO 98/24884 and WO 01/14424. Preferably,the mice will be 6-16 weeks of age upon the first infusion. For example,a purified preparation of lymphoblastoid IFN (25-100 μg), prepared bytreating a lymphoblastoid cell line with virus such that the cell lineproduces an IFN alpha preparation containing multiple IFN alpha subtypes(but not IFN omega) can be used to immunize the human Ig miceintraperitoneally. Alternatively, mixtures of recombinant forms of IFNalpha subtypes can be used as the immunogen.

Detailed procedures to generate fully human monoclonal antibodies to IFNalpha are described in Example 1 below. Cumulative experience withvarious antigens has shown that the transgenic mice respond wheninitially immunized intraperitoneally (IP) with antigen in completeFreund's adjuvant, followed by every other week IP immunizations (up toa total of 6) with antigen in incomplete Freund's adjuvant. However,adjuvants other than Freund's are also found to be effective. Inaddition, whole cells in the absence of adjuvant are found to be highlyimmunogenic. The immune response can be monitored over the course of theimmunization protocol with plasma samples being obtained by retroorbitalbleeds. The plasma can be screened by ELISA (as described below), andmice with sufficient titers of anti-IFN alpha human immunoglobulin canbe used for fusions. Mice can be boosted intravenously with antigen 3days before sacrifice and removal of the spleen. It is expected that 2-3fusions for each immunization may need to be performed. Between 6 and 24mice are typically immunized for each antigen. For HuMab mice, usuallyboth HCo7 and HCol2 strains are used. In addition, both HCo7 and HCo12transgene can be bred together into a single mouse having two differenthuman heavy chain transgenes (HCo7/HCo12). Alternatively oradditionally, the KM mouse strain can be used, as described in Example2.

Generation of Hybridomas Producing Human Monoclonal Antibodies of theInvention

To generate hybridomas producing human monoclonal antibodies of theinvention, splenocytes and/or lymph node cells from immunized mice canbe isolated and fused to an appropriate immortalized cell line, such asa mouse myeloma cell line. The resulting hybridomas can be screened forthe production of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice can be fused toone-sixth the number of P3×63-Ag8.653 nonsecreting mouse myeloma cells(ATCC, CRL 1580) with 50% PEG. Cells are plated at approximately 2×10⁵in flat bottom microtiter plate, followed by a two week incubation inselective medium containing 20% fetal Clone Serum, 18% “653” conditionedmedia, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mMHEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/mlstreptomycin, 50 mg/ml gentamycin and 1×HAT (Sigma; the HAT is added 24hours after the fusion). After approximately two weeks, cells can becultured in medium in which the HAT is replaced with HT. Individualwells can then be screened by ELISA for human monoclonal IgM and IgGantibodies. Once extensive hybridoma growth occurs, medium can beobserved usually after 10-14 days. The antibody secreting hybridomas canbe replated, screened again, and if still positive for human IgG, themonoclonal antibodies can be subcloned at least twice by limitingdilution. The stable subclones can then be cultured in vitro to generatesmall amounts of antibody in tissue culture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD280using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

Generation of Transfectomas Producing Monoclonal Antibodies of theInvention

Antibodies of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(e.g., Morrison, S. (1985) Science 229:1202).

For example, to express the antibodies, or antibody fragments thereof,DNAs encoding partial or full-length light and heavy chains, can beobtained by standard molecular biology techniques (e.g., PCRamplification or cDNA cloning using a hybridoma that expresses theantibody of interest) and the DNAs can be inserted into expressionvectors such that the genes are operatively linked to transcriptionaland translational control sequences. In this context, the term“operatively linked” is intended to mean that an antibody gene isligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g., ligation of complementary restrictionsites on the antibody gene fragment and vector, or blunt end ligation ifno restriction sites are present). The light and heavy chain variableregions of the antibodies described herein can be used to createfull-length antibody genes of any antibody isotype by inserting theminto expression vectors already encoding heavy chain constant and lightchain constant regions of the desired isotype such that the V_(H)segment is operatively linked to the C_(H) segment(s) within the vectorand the V_(L) segment is operatively linked to the C_(L) segment withinthe vector. Additionally or alternatively, the recombinant expressionvector can encode a signal peptide that facilitates secretion of theantibody chain from a host cell. The antibody chain gene can be clonedinto the vector such that the signal peptide is linked in-frame to theamino terminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel (GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)). It will be appreciated by those skilled in theart that the design of the expression vector, including the selection ofregulatory sequences, may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., theadenovirus major late promoter (AdMLP) and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or β-globin promoter. Still further, regulatory elementscomposed of sequences from different sources, such as the SRα promotersystem, which contains sequences from the SV40 early promoter and thelong terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.et al. (1988) Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Prokaryoticexpression of antibody genes has been reported to be ineffective forproduction of high yields of active antibody (Boss, M. A. and Wood, C.R. (1985) Immunology Today 6:12-13).

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular,for use with NSO myeloma cells, another preferred expression system isthe GS gene expression system disclosed in WO 87/04462, WO 89/01036 andEP 338,841. When recombinant expression vectors encoding antibody genesare introduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more preferably,secretion of the antibody into the culture medium in which the hostcells are grown. Antibodies can be recovered from the culture mediumusing standard protein purification methods.

Characterization of Antibody Binding to Antigen

Antibodies of the invention can be tested for binding to IFN alpha by,for example, standard ELISA or by Biacore analysis. Briefly, for ELISAs,microtiter plates are coated with IFN alpha (e.g., the recombinant formof different IFN alpha subtypes, or leukocyte or lymphoblastoid IFN) at0.25 μg/ml in PBS, and then blocked with 5% bovine serum albumin in PBS.Dilutions of antibody (e.g., dilutions of plasma from IFNalpha-immunized mice) are added to each well and incubated for 1-2 hoursat 37° C. The plates are washed with PBS/Tween and then incubated withsecondary reagent (e.g., for human antibodies, a goat-anti-human IgGFc-specific polyclonal reagent) conjugated to alkaline phosphatase for 1hour at 37° C. After washing, the plates are developed with pNPPsubstrate (1 mg/ml), and analyzed at OD of 405-650. Preferably, micewhich develop the highest titers will be used for fusions.

An ELISA assay as described above can also be used to screen forhybridomas that show positive reactivity with IFN alpha immunogen.Hybridomas that bind with high avidity to IFN alpha are subcloned andfurther characterized. One clone from each hybridoma, which retains thereactivity of the parent cells (by ELISA), can be chosen for making a5-10 vial cell bank stored at −140° C., and for antibody purification.

To purify anti-IFN alpha antibodies, selected hybridomas can be grown intwo-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD₂₈₀using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

To determine if the selected anti-IFN alpha monoclonal antibodies bindto unique epitopes, each antibody can be biotinylated using commerciallyavailable reagents (Pierce, Rockford, Ill.). Competition studies usingunlabeled monoclonal antibodies and biotinylated monoclonal antibodiescan be performed using IFN alpha coated-ELISA plates as described above.Biotinylated mAb binding can be detected with a strep-avidin-alkalinephosphatase probe.

To determine the isotype of purified antibodies, isotype ELISAs can beperformed using reagents specific for antibodies of a particularisotype. For example, to determine the isotype of a human monoclonalantibody, wells of microtiter plates can be coated with 1 μg/ml ofanti-human immunoglobulin overnight at 4° C. After blocking with 1% BSA,the plates are reacted with 1 μg/ml or less of test monoclonalantibodies or purified isotype controls, at ambient temperature for oneto two hours. The wells can then be reacted with either human IgG1 orhuman IgM-specific alkaline phosphatase-conjugated probes. Plates aredeveloped and analyzed as described above.

Anti-IFN alpha human IgGs can be further tested for reactivity with IFNalpha antigen by Western blotting. Briefly, cell extracts from cellsexpressing IFN alpha can be prepared and subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis. After electrophoresis, theseparated antigens are transferred to nitrocellulose membranes, blockedwith 10% fetal calf serum, and probed with the monoclonal antibodies tobe tested. Human IgG binding can be detected using anti-human IgGalkaline phosphatase and developed with BCIP/NBT substrate tablets(Sigma Chem. Co., St. Louis, Mo.).

Immunoconjugates

In another aspect, the present invention features an anti-IFN alphaantibody, or a fragment thereof, conjugated to a therapeutic moiety,such as a cytotoxin, a drug (e.g., an immunosuppressant) or aradiotoxin. Such conjugates are referred to herein as“immunoconjugates”. Immunoconjugates that include one or more cytotoxinsare referred to as “immunotoxins.” A cytotoxin or cytotoxic agentincludes any agent that is detrimental to (e.g., kills) cells. Examplesinclude taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents also include, for example,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Other preferred examples of therapeutic cytotoxins that can beconjugated to an antibody of the invention include duocarmycins,calicheamicins, maytansines and auristatins, and derivatives thereof. Anexample of a calicheamicin antibody conjugate is commercially available(Mylotarg™; Wyeth-Ayerst).

Cytoxins can be conjugated to antibodies of the invention using linkertechnology available in the art. Examples of linker types that have beenused to conjugate a cytotoxin to an antibody include, but are notlimited to, hydrazones, thioethers, esters, disulfides andpeptide-containing linkers. A linker can be chosen that is, for example,susceptible to cleavage by low pH within the lysosomal compartment orsusceptible to cleavage by proteases, such as proteases preferentiallyexpressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).

For further discussion of types of cytotoxins, linkers and methods forconjugating therapeutic agents to antibodies, see also Saito, G. et al.(2003) Adv. Drug Deliv. Rev. 55:199-215; Trail, P. A. et al. (2003)Cancer Immunol. Immunother. 52:328-337; Payne, G. (2003) Cancer Cell3:207-212; Allen, T. M. (2002) Nat. Rev. Cancer 2:750-763; Pastan, I.and Kreitman, R. J. (2002) Curr. Opin. Investig. Drugs 3:1089-1091;Senter, P. D. and Springer, C. J. (2001) Adv. Drug Deliv. Rev.53:247-264.

Antibodies of the present invention also can be conjugated to aradioactive isotope to generate cytotoxic radiopharmaceuticals, alsoreferred to as radioimmunoconjugates. Examples of radioactive isotopesthat can be conjugated to antibodies for use diagnostically ortherapeutically include, but are not limited to, iodine¹³¹, indium¹¹¹,yttrium⁹⁰ and lutetium¹⁷⁷. Method for preparing radioimmunconjugates areestablished in the art. Examples of radioimmunoconjugates arecommercially available, including Zevalin™ (IDEC Pharmaceuticals) andBexxar™ (Corixa Pharmaceuticals), and similar methods can be used toprepare radioimmunoconjugates using the antibodies of the invention.

The antibody conjugates of the invention can be used to modify a givenbiological response, and the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

Bispecific Molecules

In another aspect, the present invention features bispecific moleculescomprising an anti-IFN alpha antibody, or a fragment thereof, of theinvention. An antibody of the invention, or antigen-binding portionsthereof, can be derivatized or linked to another functional molecule,e.g., another peptide or protein (e.g., another antibody or ligand for areceptor) to generate a bispecific molecule that binds to at least twodifferent binding sites or target molecules. The antibody of theinvention may in fact be derivatized or linked to more than one otherfunctional molecule to generate multispecific molecules that bind tomore than two different binding sites and/or target molecules; suchmultispecific molecules are also intended to be encompassed by the term“bispecific molecule” as used herein. To create a bispecific molecule ofthe invention, an antibody of the invention can be functionally linked(e.g., by chemical coupling, genetic fusion, noncovalent association orotherwise) to one or more other binding molecules, such as anotherantibody, antibody fragment, peptide or binding mimetic, such that abispecific molecule results.

Accordingly, the present invention includes bispecific moleculescomprising at least one first binding specificity for IFN alpha and asecond binding specificity for a second target epitope. In a particularembodiment of the invention, the second target epitope is an Fcreceptor, e.g., human FcγRI (CD64) or a human Fcα receptor (CD89).Therefore, the invention includes bispecific molecules capable ofbinding both to FcγR, FcαR or FcεR expressing effector cells (e.g.,monocytes, macrophages or polymorphonuclear cells (PMNs)), and to targetcells expressing IFN alpha. These bispecific molecules target IFN alphaexpressing cells to effector cell and trigger Fc receptor-mediatedeffector cell activities, such as phagocytosis of an IFN alphaexpressing cells, antibody dependent cell-mediated cytotoxicity (ADCC),cytokine release, or generation of superoxide anion.

In an embodiment of the invention in which the bispecific molecule ismultispecific, the molecule can further include a third bindingspecificity, in addition to an anti-Fc binding specificity and ananti-IFN alpha binding specificity. In one embodiment, the third bindingspecificity is an anti-enhancement factor (EF) portion, e.g., a moleculewhich binds to a surface protein involved in cytotoxic activity andthereby increases the immune response against the target cell. The“anti-enhancement factor portion” can be an antibody, functionalantibody fragment or a ligand that binds to a given molecule, e.g., anantigen or a receptor, and thereby results in an enhancement of theeffect of the binding determinants for the F_(C) receptor or target cellantigen. The “anti-enhancement factor portion” can bind an F_(C)receptor or a target cell antigen. Alternatively, the anti-enhancementfactor portion can bind to an entity that is different from the entityto which the first and second binding specificities bind. For example,the anti-enhancement factor portion can bind a cytotoxic T-cell (e.g.via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell thatresults in an increased immune response against the target cell).

In one embodiment, the bispecific molecules of the invention comprise asa binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chainFv. The antibody may also be a light chain or heavy chain dimer, or anyminimal fragment thereof such as a Fv or a single chain construct asdescribed in Ladner et al. U.S. Pat. No. 4,946,778, the contents ofwhich is expressly incorporated by reference.

In one embodiment, the binding specificity for an Fey receptor isprovided by a monoclonal antibody, the binding of which is not blockedby human immunoglobulin G (IgG). As used herein, the term “IgG receptor”refers to any of the eight γ-chain genes located on chromosome 1. Thesegenes encode a total of twelve transmembrane or soluble receptorisoforms which are grouped into three Fey receptor classes: FcγRI(CD64), FcγRII(CD32), and FcγRIII (CD16). In one preferred embodiment,the Fcγ receptor a human high affinity FcγRI. The human FcγRI is a 72kDa molecule, which shows high affinity for monomeric IgG (10⁸-10⁹M⁻¹).

The production and characterization of certain preferred anti-Fcγmonoclonal antibodies are described by Fanger et al. in PCT PublicationWO 88/00052 and in U.S. Pat. No. 4,954,617, the teachings of which arefully incorporated by reference herein. These antibodies bind to anepitope of FcγRI, FcγRII or FcγRIII at a site which is distinct from theFey binding site of the receptor and, thus, their binding is not blockedsubstantially by physiological levels of IgG. Specific anti-FcγRIantibodies useful in this invention are mAb 22, mAb 32, mAb 44, mAb 62and mAb 197. The hybridoma producing mAb 32 is available from theAmerican Type Culture Collection, ATCC Accession No. HB9469. In otherembodiments, the anti-Fey receptor antibody is a humanized form ofmonoclonal antibody 22 (H22). The production and characterization of theH22 antibody is described in Graziano, R. F. et al. (1995) J. Immunol.155 (10): 4996-5002 and PCT Publication WO 94/10332. The H22 antibodyproducing cell line was deposited at the American Type CultureCollection under the designation HA022CL1 and has the accession no. CRL11177.

In still other preferred embodiments, the binding specificity for an Fcreceptor is provided by an antibody that binds to a human IgA receptor,e.g., an Fc-alpha receptor (FcαRI (CD89)), the binding of which ispreferably not blocked by human immunoglobulin A (IgA). The term “IgAreceptor” is intended to include the gene product of one α-gene (FcαRI)located on chromosome 19. This gene is known to encode severalalternatively spliced transmembrane isoforms of 55 to 110 kDa. FcαRI(CD89) is constitutively expressed on monocytes/macrophages,eosinophilic and neutrophilic granulocytes, but not on non-effector cellpopulations. FcαRI has medium affinity (≈5×10⁷ M⁻¹) for both IgA1 andIgA2, which is increased upon exposure to cytokines such as G-CSF orGM-CSF (Morton, H. C. et al. (1996) Critical Reviews in Immunology16:423-440). Four FcαRI-specific monoclonal antibodies, identified asA3, A59, A62 and A77, which bind FcαRI outside the IgA ligand bindingdomain, have been described (Monteiro, R. C. et al. (1992) J. Immunol.148:1764).

FcαRI and FcγRI are preferred trigger receptors for use in thebispecific molecules of the invention because they are (1) expressedprimarily on immune effector cells, e.g., monocytes, PMNs, macrophagesand dendritic cells; (2) expressed at high levels (e.g., 5,000-100,000per cell); (3) mediators of cytotoxic activities (e.g., ADCC,phagocytosis); (4) mediate enhanced antigen presentation of antigens,including self-antigens, targeted to them.

While human monoclonal antibodies are preferred, other antibodies whichcan be employed in the bispecific molecules of the invention are murine,chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present invention can be prepared byconjugating the constituent binding specificities, e.g., the anti-FcRand anti-IFN alpha binding specificities, using methods known in thealt. For example, each binding specificity of the bispecific moleculecan be generated separately and then conjugated to one another. When thebinding specificities are proteins or peptides, a variety of coupling orcross-linking agents can be used for covalent conjugation. Examples ofcross-linking agents include protein A, carbodiimide,N-succinimidyl-5-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686;Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Othermethods include those described in Paulus (1985) Behring Ins. Mitt. No.78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie etal. (1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents areSATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford,Ill.).

When the binding specificities are antibodies, they can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly preferred embodiment, the hinge region ismodified to contain an odd number of sulfhydryl residues, preferablyone, prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)₂ or ligand x Fab fusion protein. A bispecific molecule of theinvention can be a single chain molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. No.5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat.No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S.Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No.5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest. For example, the FcR-antibody complexes can bedetected using e.g., an enzyme-linked antibody or antibody fragmentwhich recognizes and specifically binds to the antibody-FcR complexes.Alternatively, the complexes can be detected using any of a variety ofother immunoassays. For example, the antibody can be radioactivelylabeled and used in a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986,which is incorporated by reference herein). The radioactive isotope canbe detected by such means as the use of a γ counter or a scintillationcounter or by autoradiography.

Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or a combination ofmonoclonal antibodies, or antigen-binding portion(s) thereof, of thepresent invention, formulated together with a pharmaceuticallyacceptable carrier. Such compositions may include one or a combinationof (e.g., two or more different) antibodies, or immunoconjugates orbispecific molecules of the invention. For example, a pharmaceuticalcomposition of the invention can comprise a combination of antibodies(or immunoconjugates or bispecifics) that bind to different epitopes onthe target antigen or that have complementary activities.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include an anti-IFN alpha antibody of thepresent invention combined with at least one other anti-IFN alpha agent(e.g., an immunosuppressing agent).

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,immunoconjuage, or bispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

The pharmaceutical compounds of the invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

For administration of the antibody, the dosage ranges from about 0.0001to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or withinthe range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months or onceevery three to 6 months. Preferred dosage regimens for an anti-IFN alphaantibody of the invention include 1 mg/kg body weight or 3 mg/kg bodyweight via intravenous administration, with the antibody being givenusing one of the following dosing schedules: (i) every four weeks forsix dosages, then every three months; (ii) every three weeks; (iii) 3mg/kg body weight once followed by 1 mg/kg body weight every threeweeks.

In some methods, two or more monoclonal antibodies with differentbinding specificities are administered simultaneously, in which case thedosage of each antibody administered falls within the ranges indicated.Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be, for example, weekly, monthly, every threemonths or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of antibody to the target antigen in the patient.In some methods, dosage is adjusted to achieve a plasma antibodyconcentration of about 1-1000 μg/ml and in some methods about 25-300μg/ml.

Alternatively, antibody can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe patient. In general, human antibodies show the longest half life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A “therapeutically effective dosage” of an anti-IFN alpha antibody ofthe invention preferably results in a decrease in severity of diseasesymptoms, an increase in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction. For example, in the case of systemic lupus erythematosus(SLE), a therapeutically effective dose preferably prevents furtherdeterioration of physical symptoms associated with SLE, such as, forexample, pain or fatigue. A therapeutically effective dose preferablyalso prevents or delays onset of SLE, such as may be desired when earlyor preliminary signs of the disease are present. Likewise it includesdelaying chronic progression associated with SLE. Laboratory testsutilized in the diagnosis of SLE include chemistries (including themeasurement of IFN alpha levels), hematology, serology and radiology.Accordingly, any clinical or biochemical assay that monitors any of theforegoing may be used to determine whether a particular treatment is atherapeutically effective dose for treating SLE. One of ordinary skillin the art would be able to determine such amounts based on such factorsas the subject's size, the severity of the subject's symptoms, and theparticular composition or route of administration selected.

A composition of the present invention can be administered via one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for antibodies of theinvention include intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. The phrase“parenteral administration” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion.

Alternatively, an antibody of the invention can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of the invention can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;or 4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the human monoclonal antibodies of the inventioncan be formulated to ensure proper distribution in vivo. For example,the blood-brain barrier (BBB) excludes many highly hydrophiliccompounds. To ensure that the therapeutic compounds of the inventioncross the BBB (if desired), they can be formulated, for example, inliposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat.Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise oneor more moieties which are selectively transported into specific cellsor organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade(1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K.Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I.J. Fidler (1994) Immunomethods 4:273.

Uses and Methods of the Invention

Monoclonal anti-IFN alpha antibodies and related derivatives/conjugatesand compositions of the present invention have a variety of in vitro andin vivo diagnostic and therapeutic utilities. For example, theantibodies can be used to detect IFN alpha protein, either in vitro orin vitro, using standard antibody/antigen binding assays (e.g., ELISA,RIA). Furthermore, these molecules can be administered to a subject,e.g., in vivo, to treat, prevent or diagnose a variety of disorders inwhich IFN alpha plays a role. As used herein, the term “subject” isintended to include both human and nonhuman animals. Preferred subjectsinclude human patients exhibiting autoimmune disorders. The term“nonhuman animals” of the invention includes all vertebrates, e.g.,mammals and non-mammals, such as nonhuman primates, sheep, dog, cat,cow, horse, chickens, amphibians, reptiles, etc.

The antibody compositions of the invention can be used in the treatmentof autoimmune diseases, such as systemic lupus erythematosus (SLE),multiple sclerosis (MS), inflammatory bowel disease (IBD; includingCrohn's Disease, Ulcerative Colitis and Celiac's Disease), insulindependent diabetes mellitus (IDDM), psoriasis, autoimmune thyroiditis,rheumatoid arthritis (RA) and glomerulonephritis. Furthermore, theantibody compositions of the invention can be used for inhibiting orpreventing transplant rejection or in the treatment of graft versus hostdisease (GVHD).

Antibodies of the invention can be initially tested for binding activityassociated with therapeutic use in vitro. For example, compositions ofthe invention can be tested using Biacore, ELISA and flow cytometricassays described in the Examples below. Moreover, the activity of thesemolecules can be assayed, for example, by a cell proliferation assayfollowing exposure to IFN alpha, as described in the Examples below.Suitable methods for administering antibodies and compositions of thepresent invention are well known in the art, and are described furtherabove. Suitable dosages also can be determined within the skill in theart and will depend on the age and weight of the subject and theparticular drug used. Exemplary dosages are described further above.

Anti-IFN alpha antibodies of the invention also can be co-administeredwith other therapeutic agents as described above.

As noted above, for purposes of therapy, a human antibody compositionand a pharmaceutically acceptable carrier are administered to a patientin a therapeutically effective amount. A combination of an antibodycomposition and a pharmaceutically acceptable carrier is said to beadministered in a “therapeutically effective amount” if the amountadministered is physiologically significant. An agent is“physiologically significant” if its presence results in a detectablechange in the physiology of a recipient patient. A targeted therapeuticagent is “therapeutically effective” if it delivers a higher proportionof the administered dose to the intended target than accrues at thetarget upon systemic administration of the equivalent untargeted agent.

Also within the scope of the invention are kits comprising thecompositions (e.g., human antibodies, immunoconjugates and bispecificmolecules) of the invention and instructions for use. The kit canfurther contain a least one additional reagent, such as one or moreadditional human antibodies of the invention (e.g., a human antibodyhaving a complementary activity which inhibits IFN alpha activity butthat is distinct from the first human antibody).

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference.

EXAMPLES Example 1 Generation of Human Monoclonal Antibodies Against IFNAlpha Antigen:

Natural human IFNα containing multiple sub-types purified from avirally-stimulated human lymphoblastoid cell line, resulting inproduction of multiple IFN alpha subtypes but not IFN omega, was used asthe antigen.

Transgenic Transchromosornic KM Mice™:

Fully human monoclonal antibodies to IFN alpha were prepared using theKM strain of transgenic transchromosomic mice, which expresses humanantibody genes. In this mouse strain, the endogenous mouse kappa lightchain gene has been homozygously disrupted as described in Chen et al.(1993) EMBO 12:811-820 and the endogenous mouse heavy chain gene hasbeen homozygously disrupted as described in Example 1 of PCT PublicationWO 01/09187 for HuMab mice. The mouse carries a human kappa light chaintransgene, KCo5, as described in Fishwild et al. (1996) NatureBiotechnology 14:845-851. The mouse also carries a human heavy chaintranschromosome, SC20, as described in PCT Publication WO 02/43478.

KM Mouse™ Immunizations:

To generate fully human monoclonal antibodies to IFN alpha, KM Mice™were immunized with natural human IFNα containing multiple sub-typespurified from a virally-stimulated human lymphoblastoid cell line.General immunization schemes are described in Lonberg, N. et al (1994)Nature 368: 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851 and PCT Publication WO 98/24884. The mice were 6-16 weeks of ageupon the first infusion of antigen. A purified natural preparation(25-100 μg) of IFN alpha antigen (i.e., purified from virally stimulatedlymphoblastoid cells) was used to immunize the KM Mice™ intraperitonealy(IP) or subcutaneously (Sc).

Transgenic transchromosomic mice were immunized intraperitonealy (IP) orsubcutaneously (Sc) with antigen in complete Freund's adjuvant twice,followed by 2-4 weeks IP immunization (up to a total of 8 immunizations)with the antigen in incomplete Freund's adjuvant. The immune responsewas monitored by retroorbital bleeds. The plasma was screened by ELISA(as described below), and mice with sufficient titers of anti-IFNα humanimmunogolobulin were used for fusions. Mice were boosted intravenouslywith antigen 3 and 2 days before sacrifice and removal of the spleen.

Selection of KM Mice™ Producing Anti-IFNα Antibodies:

To select KM Mice™ producing antibodies that bound IFNα, sera fromimmunized mice were tested by ELISA as described by Fishwild, D. et al.(1996). Briefly, microtiter plates were coated with purified naturalIFNα from lymphoblastoid cells at 1-2 μg/ml in PBS, 50 μl/well,incubated 4° C. overnight then blocked with 200 μl/well of 5% chickenserum in PBS/Tween (0.05%). Dilutions of plasma from IFNα immunized micewere added to each well and incubated for 1-2 hours at ambienttemperature. The plates were washed with PBS/Tween and then incubatedwith a goat-anti-human IgG Fc polyclonal antibody conjugated withhorseradish peroxidase (HRP) for 1 hour at room temperature. Afterwashing, the plates were developed with ABTS substrate (Sigma, A-1888,0.22 mg/ml) and optical density for each well was determined using aspectrophotometer set to wavelength 415 nm with a background correctionat 495 nm. Mice that developed the highest titers of anti-IFNαantibodies were used for fusions. Fusions were performed as describedbelow and hybridoma supernatants were tested for anti-IFNα activity byELISA.

Generation of Hybridomas Producing Human Monoclonal Antibodies to IFNα:

Splenocytes were isolated from KM Mice™ and fused to a mouse myelomacell line based upon standard protocols using PEG. The resultinghybridomas were then screened for the production of antigen-specificantibodies.

Single cell suspensions of splenic lymphocytes from immunized mice werefused to one-fourth the number of P3X63-Ag8.653 nonsecreting mousemyeloma cells (ATCC, CRL 1580) or SP2/0 nonsecreting mouse myeloma cells(ATCC, CRL 1581) using 50% PEG (Sigma). Cells were plated at a densityof about 1×10⁵/well in flat bottom microtiter plates and incubatedapproximately 2 weeks in selective medium containing 10% fetal bovineserum, 10% P388D1 (ATCC, CRL TIB-63) conditioned medium, 3-5% origen(IGEN) in DMEM (Mediatech, CRL 10013, with high glucose, L-glutamine andsodium pyruvate) plus 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 mg/mlgentamicin and 1×HAT (Sigma, CRL P-7185). After 1-2 weeks, cells werecultured in medium in which the HAT was replaced with HT. Individualwells were then screened by ELISA (described above) for human anti-IFNαIgG antibodies.

Conditioned medium from the antibody secreting hybridomas identified byELISA was tested in a Daudi proliferation assay (described below) forthe capacity to block the anti-proliferative effects of IFNα. Thehybridomas with highest neutralizing activity in the Daudi assay screenwere subcloned at least twice by limiting dilution. The resulting stablesubclones were then cultured in vitro to generate small amounts ofmonoclonal antibody in tissue culture medium. The Daudi proliferationassay screen was repeated to confirm the activity of the sub-clones. Thesub-clones with highest activity in the Daudi assay were scaled up toproduce sufficient conditioned medium (typically 1 L) for purificationof monoclonal anti-IFNα for further characterization.

Screen of Hybridomas for Neutralizing anti-IFNα Antibody: DaudiProliferation Assay:

Interferon alpha inhibits the proliferation of Daudi (Burkitts lymphoma,ATCC # CCL-213) cells in a dose dependant manner. A neutralizingantibody, which blocks interferon binding to its receptor, will restoreproliferation. Dose response curves for the anti-proliferative effectsof natural lymphoblastoid IFNα on Daudi were determined and aconcentration sufficient to inhibit Daudi growth by 50% (EC50) wascalculated.

Hybridoma conditioned medium was mixed with Daudi cells in culturemedium (RPMI 1640 supplemented with 10% FCS, 1×2-ME, L-glutamine andpenicillin streptomycin) with and without the addition of IFNα in a 96well, flat-bottomed cell culture plate. The final mixture of reagentswas as follows: 1×10⁴ Daudi cells+10% hybridoma supernate+/−IFNα at EC50per 100 ul/well. The cells were incubated at 37° C., 5% CO₂, 72 hrs.Proliferation was assayed with the addition of MTS (Promega), 20 ul/welland O.D. at 490 nm was read following a further 3 hrs incubation. Theviable cell number was proportional to the O.D. reading. PercentageDaudi inhibition was calculated for hybridoma supernate+IFNα relative tohybridoma supernate alone and compared to a media control with andwithout IFNα. Hybridomas were rank ordered according to the potency ofIFNα blockade and the most active neutralizing hybridomas were selectedfor sub-cloning.

Hybridoma clones 13H5, 13H7 and 7H9 were selected for further analysis.

Example 2 Structural Characterization of Human Monoclonal Antibodies13H5, 13H7 and 7H9

The cDNA sequences encoding the heavy and light chain variable regionsof the 13H5, 13H7, and 7H9 monoclonal antibodies were obtained from the13H5, 13H7, and 7H9 hybridomas, respectively, using standard PCRtechniques and were sequenced using standard DNA sequencing techniques.

The nucleotide and amino acid sequences of the heavy chain variableregion of 13H5 are shown in FIG. 1A and in SEQ ID NO: 25 and 19,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 13H5 are shown in FIG. 1B and in SEQ ID NO: 28 and 22,respectively.

Comparison of the 13H5 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 13H5 heavy chain utilizes a V_(H) segment from human germline VH1-18, an undetermined D segment, and a J_(H) segment from human germlineJ_(H) 4b. The alignment of the 13H5 V_(H) sequence to the germline VH1-18 sequence is shown in FIG. 4. Further analysis of the 13H5 V_(H)sequence using the Kabat system of CDR region determination led to thedelineation of the heavy chain CDR1, CDR2 and CD3 regions as shown inFIGS. 1A and 4, and in SEQ ID NOs: 1, 4 and 7, respectively.

Comparison of the 13H5 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 13H5 light chain utilizes a V_(L) segment from human germline VK A27and a JK segment from human germline JK 1. The alignment of the 13H5V_(L) sequence to the germline VK A27 sequence is shown in FIG. 6.Further analysis of the 13H5 V_(L) sequence using the Kabat system ofCDR region determination led to the delineation of the light chain CDR1,CDR2 and CD3 regions as shown in FIGS. 1B and 6, and in SEQ ID NOs:10,13 and 16, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 13H7 are shown in FIG. 2A and in SEQ ID NO: 26 and 20,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 13H7 are shown in FIG. 2B and in SEQ ID NO: 29 and 23,respectively.

Comparison of the 13H7 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 13H7 heavy chain utilizes a V_(H) segment from human germline VH4-61, a D segment from human germline 3-10, and a J_(H) segment fromhuman germline J_(H) 4b. The alignment of the 13H7 V_(H) sequence to thegermline VH 4-61 sequence is shown in FIG. 5. Further analysis of the13H7 V_(H) sequence using the Kabat system of CDR region determinationled to the delineation of the heavy chain CDR1, CDR2 and CD3 regions asshown in FIGS. 2A and 5, and in SEQ ID NOs: 2, 5 and 8, respectively.

Comparison of the 13H7 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 13H7 light chain utilizes a V_(L) segment from human germline VK A27and a JK segment from human germline JK 2. The alignment of the 13H7V_(L) sequence to the germline VK A27 sequence is shown in FIG. 6.Further analysis of the 13H7 V_(L) sequence using the Kabat system ofCDR region determination led to the delineation of the light chain CDR1,CDR2 and CD3 regions as shown in FIGS. 2B and 6, and in SEQ ID NOs:11,14 and 17, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 7H9 are shown in FIG. 3A and in SEQ ID NO: 27 and 21,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 7H9 are shown in FIG. 3B and in SEQ ID NO: 30 and 24,respectively.

Comparison of the 7H9 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 7H9 heavy chain utilizes a V_(H) segment from human germline VH1-18, a D segment from human germline 6-6, and a J_(H) segment fromhuman germline J_(H) 4b. The alignment of the 7H9 V_(H) sequence to thegermline VH 1-18 sequence is shown in FIG. 4. Further analysis of the7H9 V_(H) sequence using the Kabat system of CDR region determinationled to the delineation of the heavy chain CDR1, CDR2 and CD3 regions asshown in FIGS. 3A and 4, and in SEQ ID NOs: 3, 6 and 9, respectively.

Comparison of the 7H9 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 7H9 light chain utilizes a V_(L) segment from human germline VK A27and a JK segment from human germline JK 1. The alignment of the 7H9V_(L) sequence to the germline VK A27 sequence is shown in FIG. 6.Further analysis of the 7H9 V_(L) sequence using the Kabat system of CDRregion determination led to the delineation of the light chain CDR1,CDR2 and CD3 regions as shown in FIGS. 3B and 6, and in SEQ ID NOs:12,15 and 18, respectively.

Example 3 Anti-IFN Alpha Human Monoclonal Antibodies Inhibit theBiological Activity of Multiple Interferon Alpha Subtypes

As described in Example 1, interferon alpha inhibits the proliferationof Daudi (Burkitts lymphoma, ATCC # CCL-213) cells in a dose dependantmanner. A neutralizing antibody, which blocks interferon binding to itsreceptor, will restore proliferation. Using this cell proliferationassay, the specificity of the purified human anti-IFN alpha antibodieswas examined by testing for blockade of natural lymphoblastoid IFNα,natural leukocyte interferon, 13 recombinant IFN alpha subtypes, IFNbeta and IFN omega.

Daudi cells were grown in culture medium (RPMI 1640 supplemented with10% FCS, 1×2-ME, L-glutamine and penicillin streptomycin) with andwithout the addition of IFNα in a 96 well, flat-bottomed cell cultureplate. Each type I interferon tested was assayed at EC₅₀ and mixed witha 2-fold serial titration of each antibody, typically from 50 ug/ml (312nM) through 381 pg/ml (2.4 pM). The antibody/IFN mixture was added toDaudi cells in a 96-well bottomed plate to a final density of 1×10⁴Daudi cells/100 ul/well and incubated at 37° C., 5% CO₂, 72 hrs.Proliferation was assayed with the addition of MTS (Promega), 20ul/well, and O.D. at 490 nm was measured following a further 3 hourincubation. The viable cell number was proportional to the O.D. reading.Percentage blockade of interferon was calculated relative to Daudiproliferation in the absence of IFN (=100% blockade) and in the presenceof IFN alone (=0% blockade). Antibodies were scored according to thedegree of blockade, resulting in a profile of IFNα subtype specificityfor each antibody tested. An EC₅₀ was derived with PRISM™ software usingnon-linear regression; sigmoidal dose response; variable slope curvefit. The results demonstrated that the human anti-IFN alpha antibody13H5 inhibits the action of multiple interferon alpha subtypes,particularly, IFNα 6, 2b, 2a, 1, 16, 10, 8, 5 and 14, but not IFNα 21,IFNβ or IFNω. 13H5 is a low level inhibitor of IFN alpha subtypes 17, 7and 4. The EC₅₀ values and % blockade of interferon are shown in table1, below.

TABLE 1 Antibody Inhibition of Multiple IFN Alpha Subtypes 13H5 IFNBlockade IFN EC50 1000x Lymphoblastoid IFN 127 pM 82% IFNα 6 208 pM 95%IFNα 2b 432 pM 80% IFNα 2a 448 pM 95% IFNα 1 4.6 nM 68% Leukocyte IFN5.5 nM 70% IFNα 16 6.8 nM 80% IFNα 10 19.6 nM 40% IFNα 8 26 nM 37% IFNα5 56 nM 47% IFNα 14 70 nM 34% IFNα 17 110 nM 13% IFNα 7 >300 nM 15% IFNα4 >300 nM  7% IFNα 21 >300 nM NS IFN-beta >300 nM NS IFN-omega >300 nMNS NS = not significant

Example 4 Inhibition of IFN Alpha Induction of Cell Surface Markers byAnti-IFN Alpha Antibodies

The addition of IFN alpha 2b to cell culture media is known to inducethe expression of the cell surface markers CD38 and MHC Class I onnormal peripheral blood mononuclear cells (PBMNC). The activity of humananti-IFN alpha antibody 13H5 was tested for inhibition of interferoninduced cell surface marker expression on cultures of primary humancells and assayed by FACS analysis.

The anti-IFNα monoclonal antibody 13H5 and isotype controls were dilutedto 20 ug/ml each in PBMNC culture medium (RPMI 1640+10% FBS+1% humanserum). Antibody was dispensed 1.5 ml/well into T25 vented cap cultureflasks and mixed with an equal volume of either 400 iu/ml leukocyte IFN,IFN alpha 2b or IFN ω, diluted in culture medium or with medium alone.PBMNC were isolated from normal human blood using heparin coatedVacutainer® CPT™ tubes according to manufacturer recommendations (BectonDickinson & Co). Cells were resuspended in culture medium (RPMI 1640+10%FBS+1% human serum) to 2×10⁶ cells/ml and were added in equal volume tothe Ab/IFN mixtures such that the final assay contains; 6×10⁶ PBMNC+5ug/ml Ab+/−100 iu/ml IFN per 6 ml medium. Flasks were incubated at 37°C., 5% CO₂ for 24 or 48 hrs.

Conditioned medium was harvested from each flask and suspension cellswere recovered by centrifugation at 1000 rpm on a Sorvall RTH-750 rotor.The pelleted cells were retained on ice and supernate was frozen at −80°C. for ELISA. Adherent cells were recovered from the flask with a PBSwash (2 ml), followed by 15 minute incubation in versene (3 ml). Theflask was scraped at the end of the versene incubation and the flask wasfinally rinsed with PBS wash (2 ml). Each of the PBS washes and theversene was combined with the cells recovered from conditioned mediumharvest. The pooled cell suspension was centrifuged at 1000 rpm on aSorvall RTH-750 rotor, the resulting pellet was resuspensed to 300 ul instaining buffer (PBS+0.1M EDTA+2% FBS+1% HS) and dispensed 100 ul/wellinto a V-bottom 96-well plate.

The plate was pulse-centrifuged at 2800 rpm on a Sorvall RTH-750 rotorand pelleted cells were resuspended 25 μl/well in fluorochrome labeledantibodies as follows: (1) mouse anti-MHC I-FITC+mouse anti-CD38-PE, and(2) isotype controls, mouse IgG-FITC+mouse IgG-PE. The plate wasincubated on ice for 45 minutes, protected from light. The cells werewashed three times with the addition of 200 ul staining buffer followedby pulse-celtrifugation and finally resuspended in 200 μl of 2%paraformaldehyde in PBS. Staining of monocyte cells was analyzed by flowcytometry with the Becton Dickinson FACScalibur™, gates were drawn onthe Forward Scatter vs. Side Scatter graph to remove contaminating cellsfrom the analysis. The results demonstrated that the human monoclonalantibody 13H5 inhibits leukocyte IFN and recombinant IFNα 2b inducedchanges in expression of CD38 and MHC Class I on normal PBMNC. The humanmonoclonal antibody 13H5 does not block IFNω mediated changes in thecell surface marker expression of CD38 and MHC Class I. These resultsare shown in Tables 2 and 3 below.

TABLE 2 Percent Change in IFN-Induced MHC Class I Expression on NormalPBMNC Leukocyte IFN IFN alpha 2b IFN omega Ab Treatment 100 u/ml 100u/ml (100 u/ml) No antibody 31 21 28 13H5 (5 μg/ml) −1 −1 29 Control Ig(5 μg/ml) 16 25 26

TABLE 3 Percent Change in IFN-Induced CD38 Expression on Normal PBMNCLeukocyte IFN IFN alpha 2b IFN omega Ab Treatment (100 u/ml) (100 u/ml)(100 u/ml) No antibody 774 426 782 13H5 (5 μg/ml) 195 16 760 Control Ig(5 μg/ml) 614 392 829

Example 5 Inhibition of IFN-Induction Expression of IP-10 by Anti-IFNAlpha Antibodies

The addition of IFN alpha 2b to cell culture media is known to induceIP-10 expression in normal peripheral blood mononuclear cells (PBMNC).The activity of human anti-IFN alpha antibody 13H5 was tested forinhibition of interferon induced expression of IP-10 in normal PBMNCcultures by an ELISA binding assay.

A PBMNC culture was prepared as described in Example 4, conditioned withleukocyte IFN, IFN alpha 2b, or IFN ω. Conditioned medium was analyzedfor IP-10/CXCL10 expression using a quantitative sandwich ELISA kit(Quantikine®, R&D Systems) at a 1:30 dilution according to manufacturerrecommendations. The results demonstrated that the human monoclonalantibody 13H5 inhibits leukocyte IFN and recombinant IFNα 2b inducedexpression of IP-10 in normal PBMNC culture but does not block IFNωinduced IP-10 expression. These results are shown in Table 4.

TABLE 4 Antibody Inhibition of in IFN-Induced IP-10 Expression on NormalPBMNC No Leukocyte IFN IFN alpha 2b IFN omega Ab Treatment IFN (100u/ml) (100 u/ml) (100 u/ml) No antibody 907 2665 2739 2904 13H5 (5μg/ml) 946 1765 1262 3862 Control Ig (5 μg/ml) 838 3512 3117 3960

Example 6 Affinity Characterization of Anti-IFN Alpha Human MonoclonalAntibody

In this example, the monoclonal antibody 13H5 was examined for bindingaffinity of recombinant IFN alpha 2a and IFN alpha 2b using Biacoreanalysis.

Purified antibodies at 10 ug/ml, were captured on a CM5 chip coated withProt-G. Concentrations of antigen from 80 nM to 10 nM in HBS-EP runningbuffer was passed over the chip at a rate of 25 ul/min. The associationtime allowed was 5 minutes, followed by a 10 minute dissociation period.Background and non-specific binding of antigen to both the chip andantibodies was eliminated by detecting the binding to surface withcaptured isotype control human-IgG (Sigma) and buffer. Regeneration ofthe chip was achieved with a flow rate of 100 ul/min for 0.4 minutesusing 20 mM NaOH+400 mM NaCl. The association and dissociation curveswere fit to a Langmuir binding model using BlAevaluation software(Biacore AB). The results are shown below in Table 5.

TABLE 5 Binding Characteristics of Monoclonal Antibody 13H5 IFN AlphaSubtype K_(D) K_(on) K_(off) IFN Alpha 2a 1.0 × 10⁻¹⁰ M 3.3 × 10⁻⁵ 1/Ms3.5 × 10⁻⁵ 1/Ms IFN Alpha 2b 1.0 × 10⁻¹⁰ M 5.1 × 10⁻⁵ 1/Ms 5.3 × 10⁻⁵1/Ms

Example 7 Antibody Inhibition of SLE Plasma Mediated Dendritic CellDevelopment

SLE plasma induces dendritic cell development from normal humanmonocytes. In this example, purified monoclonal human anti-IFN alphaantibodies were tested for inhibition of dendritic cell development, asassessed by the ability of the antibodies to inhibit the induction ofthe cell surface markers CD38, MHC Class I and CD123 by SLE plasma.

A 25 ml buffy coat was diluted four fold with PBS. The sample wasseparated into 4×50 ml conical tubes, and 15 ml of lymphocyte separationmedium (ICN Biomedicals) was layered underneath. Following a 30-minutespin at 500×g, the buffy layer containing the PBMCs was removed andwashed with PBS. Cells were resuspended in culture media at 4×10⁶cells/ml. Monocytes were isolated by incubating PBMC (2.0×10⁷ cells/5ml/25 cm² flask) for 1.5 hrs at 37° C. in culture medium and thenwashing away non-adherent cells twice. Following the second wash thecells were cultured in media containing 1% heat inactivated human serum.Twenty five percent SLE patient plasma plus/minus neutralizingantibodies and isotype controls (30 ug/ml) were added to the cultureflasks; IFN alpha 2b (100 & 10 iu/ml) plus 25% normal human plasma wasused as a positive control for marker induction. Flasks were incubatedat 37° C., 5% CO₂ for three to seven days. Dendritic cells were thenrecovered from conditioned medium, with PBS and versene treatment ifnecessary, before being stained as described for blockade of markerinduction in PBMNC culture (as described in Example 4 above). Stainingof dendritic cells was analyzed by flow cytometry with the BectonDickinson FACScalibur™. Gates were drawn on the Forward Scatter vs. SideScatter graph to remove contaminating cells from the analysis. Theanti-IFN alpha human monoclonal antibody 13H5 inhibits the IFN alphadependent process of dendritic cell development, as demonstrated bynormalized expression of cell surface markers MHC Class I, CD38, andCD123 in the presence of 13H5. The results are shown below in Table 6,wherein (A), (13), (C) & (D) summarize results for four representativeSLE donor samples.

TABLE 6 Inhibition of Dendritic Cell Maturation Donor Plasma DonorPlasma 40 (13 iu/ml IFN) 39 (19 iu/ml IFN) (A) Culture Cond MHC I CD123CD38 (B) Culture Cond MHC I CD123 CD38 0 IFN 148.34 14.22 39.78 0 IFN248.83 18.63 32.69 10 iu/ml IFNa 2b 199.84 18.92 44.18 10 iu/ml IFNa 2b331.82 21.42 63.23 100 iu/ml IFNa 2b 229.05 26.27 63.36 100 iu/ml IFNa2b 430.87 30.56 60.61 0 Ab 206.02 22 46.78 0 Ab 443.21 17.53 44.87 13H5144.92 13.67 35.11 13H5 330.59 14.18 20.56 Control IgG 193.52 21.5 62.04Control IgG 432.43 17.88 39.33 Donor Plasma Donor Plasma 36 59 (75 iu/mlIFN) (′C) Culture Cond MHC I CD123 CD38 (D) Culture Cond MHC I CD123CD38 0 IFN 358.88 15.25 45.75 0 IFN 228.96 10.5 58 10 iu/ml IFNa 2b457.133 17.41 58.48 10 iu/ml IFNa 2b 271.19 11.95 86.49 100 iu/ml IFNa2b 496.32 20.63 64.55 100 iu/ml IFNa 2b 293.99 12.73 112.49 0 Ab 488.5828.92 88.31 0 Ab 202.04 14.74 61.61 13H5 429.31 15.44 73.88 13H5 127.229.17 30.79 Control IgG 485.7 19.75 115.18 Control IgG 266 14.4 55.46

Example 8 Mechanism of Action of Monoclonal Antibody 13H5

In this example, several binding experiments using radiolabeled cytokineand antibody with IFNAR expressing cells were conducted in order todetermine the mechanism of action for 13H5.

In the first set of experiments, recombinant IFNα 2a was radio-iodinatedwith a specific activity of 29.3 Ci/mmole (Pierce IODO-GEN® tubes) andwas determined to specifically bind Daudi cells with a K_(D) ofapproximately 1 nM. To examine competition binding of this ligand tocells, glass fiber plates were blocked with 200 μl/well milk bufferovernight at 4° C. Daudi cells were dispensed at 2×10⁶ cells/well inRPMI 1640 medium and were mixed with ¹²⁵I-IFNα (2 nM), plus a 3-folddilution series of competitor, either 13H5, an isotype control antibodyor unlabelled IFNα (30 nM to 14 pM). The plate was incubated 2 hours at4° C. on a shaker before being washed with RPMI and air-dried. Thefilters were transferred to glass tubes and analyzed for radioactivity.

Representative results from several experiments are shown in FIG. 7.Unlabeled ligand was used as a positive control and was observed tospecifically block ¹²⁵I-IFNα binding with an IC₅₀ of approximately 0.5nM. The 13H5 antibody, however, did not block binding of iodinatedligand but was instead observed to enhance the radioactive signalassociated with treated cells, contrasting with the behavior of theisotype control antibody, which had no effect on ¹²⁵I-IFNα binding tocells. This result indicates that 13H5 has a non-competitive mechanismof action and neutralizes biological activity by blockade of signalingbut not by blockade of ligand binding.

The above result also suggested that 13H5 may become associated with thecell surface in the presence of IFNα. Since each 13H5 molecule has thecapacity to bind two IFNα molecules, it is possible that these eventswould also result in a second ligand being linked to the cell membrane.This hypothesis is supported by the observation that cell-associatedradioactivity was enhanced approximately 2-fold at concentrations ofantibody and ligand consistent with a 1:1 ratio of IFNα to 13H5 bindingsites.

To further examine the mechanism of action of 13H5, the binding of theantibody to Daudi cells was assayed using radiolabeled antibody in thepresence or absence of IFNα 2a. The cytokine was used at a concentration(10 nM) calculated to saturate IFNAR binding based upon earlier bindingstudies. The 13H5 antibody was radio-iodinated with a specific activityof 414 Ci/mmole (Pierce IODO-GEN® tubes). To examine antibody binding tocells, glass fiber plates were blocked with 200 μl/well milk bufferovernight at 4° C. Daudi cells were dispensed at 2×10⁶ cells/well inRPMI 1640 medium and were mixed with a 2-fold diluation series of¹²⁵-13H5 (20 nM to 20 pM), plus/minus IFNα 2a (10 nM). The plate wasincubated 2 hours at 4° C. on a shaker before being washed with RPMI andair-dried. The filters were transferred to glass tubes and analyzed forradioactivity. CPM values measured for ¹²⁵-13H5 binding alone weresubtracted from those measured in the presence of IFNα 2a in order todetermine IFNα 2a dependent binding. Representative results from severalexperiments are shown in FIG. 8. The results showed dose dependantsaturable binding of ¹²⁵-13H5 to Daudi cells in the presence of IFNα 2abut negligible binding with ¹²⁵-13H5 alone. The specific IFNα-dependentbinding of 13H5 is represented in FIG. 8 by circles and was calculatedby subtracting CPM for antibody alone (representing non-specificbinding) from total CPM for 13H5 binding in the presence of IFN x.

Thus, in summary, the mechanism of action of 13H5 is a non-competitiveone in which the complex of IFNα bound to 13H5 is capable of binding toIFNAR on the cell surface and the biological activity of IFNα isneutralized by blockade of signaling through IFNAR.

Example 9 Antibody Dependent Cell-Mediated Cytotoxicity Assays with 13H5

Since 13H5 can associate with the cell surface in the presence of IFNα,antibody dependent cell-mediated cytotoxicity (ADCC) was investigatedusing a ⁵¹Cr-release assay. Raji cells were used as targets for lysis byfresh human mononuclear cells. Mononuclear cells were purified fromheparinized whole blood by Ficoll Hypaque density centrifugation. Targetcells were labeled with 100 μCi of ⁵¹Cr per 10⁶ cells for 1 hour priorto dispensing into U-bottom microtiter plates, 10⁴ cells per well, andcombining with effector cells (effector:target ratio=50:1) plustitrations of antibody. Following 4 hours incubation at 37° C.,supernatant conditioned medium was collected and analyzed forradioactivity. Release of radioactivity in the absence of antibody wasused as a control for background and detergent treatment of target cellswas used to determine 100% lysis. Cytotoxicity was calculated by theformula: % lysis=(experimental cpm−target leak cpm)/(detergent lysiscpm−target leak cpm)×100%. Specific lysis=% lysis with 13H5-% lysiswithout 13H5. Assays were performed in triplicate.

The results of the ADCC assay, summarized in FIG. 9, demonstrate that13H5 had no significant ADCC activity on Raji cells, either alone or inthe presence of IFNα 2b. Similarly, an isotype matched IgG displayed noactivity, whereas the positive control (Rituximab) exhibited robust dosedependent cytotoxicity. These results indicate that IFNα mediatedassociation of 13H5 with the cell surface of IFNAR expressing cells isnot sufficient to mediate ADCC.

Example 10 Examination of Stability of 13H5

The 13H5 antibody contains a potential deamidation site at Asn-55 in theCDR2 region of the heavy chain. Deamidation of asparagines residues is acommon modification of polypeptides and proteins obtained usingrecombinant DNA technology and may result in decreased biologicalactivity and/or stability, though deamidation does not always correlatewith loss of biological activity. Deamidation of asparagines to formaspartic acid (and iso-Asp) results in a change of net charge, which canbe detected by charge-based analytical methods. To examine deamidationof 13H5 under accelerated conditions (basic pH), methods for detectionof deamidated variants of Fab fragment by IEX-HPLC and capillaryisoelectric focusing (cEIF) were used.

To accelerate deamidation of 13H5, the antibody was exposed to buffer atalkaline pH. For the starting material, a 102 μl aliquot of 13H5 (at5.9. mg/ml for a total of 600 μg) was added to 498 μl of PBS and 6 μl of100× sodium azide stock (2% solution). For the time zero PBS sample, 130μl of starting material was combined with 30 μl of PBS and the samplewas placed at −20° C. until further analysis. For the time zero samplein deamidation buffers, 130 μl of starting material was combined with 15μl of 10× deamidation buffer (10% ammonium bicarbonate, pH 8.5) and 15μl of pH adjustment buffer (1M MES, pH 6.0) and placed at −20° C. untilfurther analysis. For the Day 2 sample in PBS, 130 μl of startingmaterial was combined with 30 μl of PBS and incubated at 40° C. for 48hours and then the sample was placed at −20° C. until further analysis.For the Day 2 sample under deamidation conditions, 130 μl of startingmaterial was combined with 15 μl of 10× deamidation buffer and incubatedat 40° C. for 48 hours. After 48 hours, 15 μl of pH adjustment bufferwas added and the sample was placed at −20° C. until further analysis.

To prepare the above samples for analysis, papain digestion wasperformed. Reaction conditions used were: 160 μl of sample (130 μg13H5), 3.2 μl of 50 mM cysteine and 6.5 μl of papain enzyme at 1.0 mg/mlin solution. The samples were placed at 40° C. for 4 hours and thereaction was stopped by addition of 4.8 μl of 1M iodoacetamide. Afterpapain digestion, non-reducing SDS-PAGE was performed to confirm thepresence of Fab and Fc fragments.

To perform IEX-HPLC on the samples, all samples were first dialyzedagainst water for 3 hours. Then, 50 μl of each sample was applied toHPLC with the following chromatography conditions:

-   -   Column=Dionex WCX-10 weak cation exchange column    -   “A” buffer=10 mM MES, pH 5.5    -   “B” buffer=10 mM MES, pH 5.5; 1.0 M NaCl    -   Elution=4-25% “B” over 30 minutes at 0.8 ml/min    -   Detection=UV absorbance at 280 nM        The results of IEX-HPLC analysis are summarized in Table 7        below, which shows the peak areas for deamidated Fab for time        zero and Day 2 samples under deamidation conditions:

TABLE 7 Peak Area (% peak area) (% peak Deamidated Deamidated Peak Areaarea) Total Peak Sample Fab Fab Fab Fab Area Time 0 89,244 6.131,366,233 93.87 1,455,477 Day 2 459,759 43.95 586,428 56.01 1,046,187

To perform cIEF analysis, samples were first dialyzed against water for3 hours and then applied to cIEF using standard methods of analysis. Theresults of cIEF analysis are summarized in Table 8 below, which showsthe peak areas for deamidated Fab for time zero and Day 2 samples underdeamidation conditions:

TABLE 8 Peak Area (% peak area) (% peak Deamidated Deamidated Peak Areaarea) Total Peak Sample Fab Fab Fab Fab Area Time 0 75,902 13.96 467,98786.04 543,889 Day 2 251,317 58.81 176,040 41.19 427,357

To examine the pH dependence of forced deamidation, the IEX-HPLC datafor the Day 2 sample in PBS (pH 7.0) was compared to the Day 2 sampleunder deamidation conditions. The results are summarized in Table 9below, which shows the peak areas for the deamidated Fab for Day 2 PBSand Day 2 under deamidation conditions:

TABLE 9 Peak Area (% peak area) (% peak Deamidated Deamidated Peak Areaarea) Total Peak Sample Fab Fab Fab Fab Area PBS 106,344 7.0 1,413,23393.0 1,519,577 deami- 459,759 43.95 586,428 56.01 1,046,187 datedThis data supports the existing theory of protein degradation, whichpredicts that deamidation of polypeptides via beta-aspartyl shiftmechanism occurs at an increased rate under basic pH as compared toneutral pH.

Example 11 Preparation and Characterization of 13H5 Mutants withEnhanced Stability

In this example, 13H5 mutants were prepared having an amino acidsubstitution at Asn-55 and the stability of these mutants was examined,at Day 2 under forced deamidation conditions, by cIEF analysis asdescribed in Example 10. The mutants were prepared by standardrecombinant DNA mutagenesis techniques. The sequences of the mutants atamino acid positions 55-58 of V_(H), as compared to wild type 13H5, wereas follows:

13H5 wild-type: N G N T (SEQ ID NO: 41) Mutant #1: D G N T(SEQ ID NO: 38) Mutant #2: Q G N T (SEQ ID NO: 39) Mutant #3: Q G Q T(SEQ ID NO: 40)The full-length V_(H) amino acid sequences of mutants #1, #2 and #3 areshown in SEQ ID NOs: 34, 35 and 36, respectively.

The results of the cIEF analysis are shown below in Table 10, whichshows the peak areas for deamidated Fab for the wild type and mutants atDay 2 deamidation conditions:

TABLE 10 Peak Area (% peak area) (% peak Deamidated Deamidated Peak Areaarea) Total Peak Sample Fab Fab Fab Fab Area 13H5 24,065 54.2 20,30445.8 44,369 wild type Mu- 10,382 9.7 96,584 90.3 106,966 tant 1 Mu-4,592 8.0 52,460 92.0 57,052 tant 2 Mu- 7,733 8.9 79,077 91.1 86,810tant 3The results demonstrate that each of the three Asn-55 mutants exhibitsgreater stability under forced deamidation conditions than the wild-type13H5 antibody.

SUMMARY OF SEQUENCE LISTING SEQ ID NO: SEQUENCE 1 VH CDR1 a.a. 13H5 2 VHCDR1 a.a. 13H7 3 VH CDR1 a.a. 7H9 4 VH CDR2 a.a. 13H5 5 VH CDR2 a.a.13H7 6 VH CDR2 a.a. 7H9 7 VH CDR3 a.a. 13H5 8 VH CDR3 a.a. 13H7 9 VHCDR3 a.a. 7H9 10 VK CDR1 a.a. 13H5 11 VK CDR1 a.a. 13H7 12 VK CDR1 a.a.7H9 13 VK CDR2 a.a. 13H5 14 VK CDR2 a.a. 13H7 15 VK CDR2 a.a. 7H9 16 VKCDR3 a.a. 13H5 17 VK CDR3 a.a. 13H7 18 VK CDR3 a.a. 7H9 19 VH a.a. 13H520 VH a.a. 13H7 21 VH a.a. 7H9 22 VK a.a. 13H5 23 VK a.a. 13H7 24 VKa.a. 7H9 25 VH n.t. 13H5 26 VH n.t. 13H7 27 VH n.t. 7H9 28 VK n.t. 13H529 VK n.t. 13H7 30 VK n.t. 7H9 31 VH 1-18 germline a.a. 32 VH 4-61germline a.a. 33 VK A27 germline a.a. 34 VH a.a. 13H5 N55D mut. 35 VHa.a. 13H5 N55Q mut. 36 VH a.a. 13H5 N55Q mut. N57Q mut. 37 VH a.a. 13H5G56A mut.

1. A method of treating an interferon alpha-mediated disease or disorderin a subject in need of treatment comprising administering to thesubject an effective amount of an isolated anti-interferon alphamonoclonal antibody, or an antigen binding portion thereof, which bindsan epitope on a human interferon alpha polypeptide recognized by anantibody comprising a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 19 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 22, such that theinterferon-alpha mediated disease in the subject is treated.
 2. A methodof treating an interferon alpha-mediated disease or disorder in asubject in need of treatment comprising administering to the subject aneffective amount of an isolated anti-interferon alpha monoclonalantibody, or antigen binding portion thereof, comprising: (a) a heavychain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 19, 34, 35, 36 and 37, and (b) alight chain variable region comprising the amino acid sequence of SEQ IDNO: 22, such that the interferon-alpha mediated disease in the subjectis treated.
 3. A method of treating an interferon alpha-mediated diseaseor disorder in a subject in need of treatment comprising administeringto the subject an effective amount of an isolated anti-interferon alphamonoclonal antibody, or antigen binding portion thereof, whichcomprises: (a) a heavy chain variable region CDR1 comprising SEQ ID NO:1; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 4; (c) aheavy chain variable region CDR3 comprising SEQ ID NO: 7; (d) a lightchain variable region CDR1 comprising SEQ ID NO: 10; (e) a light chainvariable region CDR2 comprising SEQ ID NO: 13; and (f) a light chainvariable region CDR3 comprising SEQ ID NO: 16, such that theinterferon-alpha mediated disease in the subject is treated.
 4. Themethod of any one of claims 1, 2, and 3, wherein the disease or disorderis selected from the group consisting of: systemic lupus erythematosus,multiple sclerosis, inflammatory bowel disease, insulin dependentdiabetes mellitus, psoriasis, autoimmune thyroiditis, rheumatoidarthritis, glomerulonephritis, transplant rejection and graft versushost disease.
 5. The method of any one of claim 2 or 3, wherein theantibody, or antigen binding portion thereof, comprises: (a) a heavychain variable region comprising the amino acid sequence of SEQ ID NO:19, and (b) a light chain variable region comprising the amino acidsequence of SEQ ID NO:
 22. 6. The method of any one of claims 1, 2, and3, wherein anti-interferon alpha monoclonal antibody, or antigen bindingportion thereof, is administered by a route selected from the groupconsisting of subcutaneously, intramuscularly, intraarterially,intradermally, and intravenously.
 7. The method of any one of claims 1,2, and 3, wherein the anti-interferon alpha monoclonal antibody, orantigen binding portion thereof, further comprises a pharmaceuticallyacceptable carrier.
 8. The method of any one of claims 1, 2, and 3,wherein the antibody, or antigen binding portion thereof, inhibitsIFN-induced surface expression of CD38 or MHC Class I on peripheralblood mononuclear cells.
 9. The method of any one of claims 1, 2, and 3,wherein the antibody, or antigen binding portion thereof, inhibitsIFN-induced expression of IP-10 by peripheral blood mononuclear cells.10. The method of any one of claims 1, 2, and 3, wherein the antibody,or antigen binding portion thereof, inhibits dendritic cell developmentmediated by systemic lupus erythematosus (SLE) plasma.
 11. The method ofany one of claims 1, 2, and 3, wherein the antibody is a human antibody.12. The method of any one of claims 1, 2, and 3, wherein the antibody isa chimeric antibody.
 13. The method of any one of claims 1, 2, and 3,wherein the antibody is a humanized antibody.
 14. The method of any oneof claims 1, 2, and 3, wherein the antigen binding portion is a Fabantibody fragment.
 15. The method of any one of claims 1, 2, and 3,wherein the antigen binding portion is a single chain antibody (scFv).16. The method of any one of claims 1, 2, and 3, wherein the subject ishuman.